Plasma kallikrein binding proteins

ABSTRACT

Plasma kallikrein binding proteins and methods of using such proteins are described.

This application is a continuation of U.S. application Ser. No.14/339,053, filed Jul. 23, 2014, which is a divisional of U.S.application Ser. No. 12/985,914, issued as U.S. Pat. No. 8,822,653,which claims priority to U.S. Application Ser. No. 61/292,614, filed onJan. 6, 2010. The contents of each of the prior applications areincorporated by reference herein in their entirety.

BACKGROUND

Plasma kallikrein is a serine protease. Prekallikrein is the precursorof plasma kallikrein.

SUMMARY

Plasma kallikrein is a serine protease component of the contact systemand a potential drug target for different inflammatory, cardiovascular,infectious (sepsis) and oncology diseases (Sainz I. M. et al., ThrombHaemost 98, 77-83, 2007). The contact system is activated by eitherfactor XIIa upon exposure to foreign or negatively charged surfaces oron endothelial cell surfaces by prolylcarboxypeptidases (FIG. 1) (SainzI. M. et al., Thromb Haemost 98, 77-83, 2007). Activation of the plasmakallikrein amplifies intrinsic coagulation via its feedback activationof factor XII and enhances inflammation via the production of theproinflammatory nonapeptide bradykinin. As the primary kininogenase inthe circulation, plasma kallikrein is largely responsible for thegeneration of bradykinin in the vasculature. A genetic deficiency in theC1-inhibitor protein (C1-INH), the major natural inhibitor of plasmakallikrein, leads to hereditary angioedema (HAE). Patients with HAEsuffer from acute attacks of painful edema often precipitated by unknowntriggers (Zuraw B. L. et al., N Engl J Med 359, 1027-1036, 2008).Through the use of pharmacological agents or genetic studies in animalmodels, the plasma kallikrein-kinin system (plasma KKS) has beenimplicated in various diseases.

Plasma kallikrein binding proteins (e.g., antibodies, e.g., inhibitoryantibodies) are useful therapeutic agents for a variety of diseases andconditions, e.g., diseases and conditions that involve plasma kallikreinactivity, due to their high potency, specificity, and prolonged serumresidency. High potency can translate to efficacy and a low drug dosage,and high specificity can reduce side effects due to the inhibition ofrelated off target serine proteases. In general, small molecule serineproteases are not as specific as antibody inhibitors. Prolonged serumresidency can permit infrequent dosing.

In some aspects, the disclosure features an isolated protein (e.g.,antibody, e.g., human antibody) that binds to the active form of plasmakallikrein (e.g., human plasma kallikrein and/or mouse plasmakallikrein), and, e.g., does not bind preplasma kallikrein (e.g., humanpreplasma kallikrein and/or mouse preplasma kallikrein).

In some embodiments, the plasma kallikrein binding protein binds thesame epitope or competes for binding with a kallikrein binding proteindescribed herein. In some embodiments, the plasma kallikrein bindingprotein binds the same epitope or competes for binding with a protein(e.g., epi-Kal2) and/or a small molecule (e.g., AEBSF) described hereinand does not bind pre-plasma kallikrein.

In some embodiments, the protein described herein is selected from thegroup consisting of M162-A04, M160-G12, M142-H08, X63-G06, X101-A01(also referred to herein as DX-2922), X81-B01, X67-D03, X67-G04,X115-B07, X115-D05, X115-E09, X115-H06, X115-A03, X115-D01, X115-F02,X115-G04, M29-D09, M145-D11, M06-D09 and M35-G04.

In some embodiments, the plasma kallikrein binding protein competes withor binds the same epitope as X81-B01 and, e.g., does not bind pre-plasmakallikrein.

In some embodiments, the plasma kallikrein binding protein competes withor binds the same epitope as X67-D03 and, e.g., does not bind pre-plasmakallikrein.

In some embodiments, the plasma kallikrein binding protein competes withor binds to the same site as X101-A01 and, e.g., does not bindpre-plasma kallikrein.

In some embodiments, the plasma kallikrein binding protein competes withor binds to the same site as M162-A04 and, e.g., does not bindpre-plasma kallikrein.

In some embodiments, the plasma kallikrein binding protein competes withor binds to the same site as X63-G06 and, e.g., does not bind pre-plasmakallikrein.

In some embodiments, the plasma kallikrein binding protein does not bindprekallikrein (e.g., human prekallikrein and/or mouse prekallikrein),but binds to the active form of plasma kallikrein (e.g., human plasmakallikrein and/or mouse plasma kallikrein).

In certain embodiments, the protein binds at or near the active site ofthe catalytic domain of plasma kallikrein, or a fragment thereof, orbinds an epitope that overlaps with the active site of plasma kallikreinand, e.g., does not bind pre-plasma kallikrein.

In some embodiments, the protein binds to one or more amino acids thatform the catalytic triad of plasma kallikrein: His434, Asp483, and/orSer578 (numbering based on the human sequence) and, e.g., does not bindpre-plasma kallikrein.

In some embodiments, the protein binds to one or more amino acids of:Ser479, Tyr563, and/or Asp585 (numbering based on the human sequence)and, e.g., does not bind pre-plasma kallikrein.

In some embodiments, the plasma kallikrein binding protein binds one ormore amino acids of: Arg551, Gln553, Tyr555, Thr558, and/or Arg560(numbering based on the human kallikrein sequence). In otherembodiments, the plasma kallikrein binding protein binds two, three,four or five (i.e., all) amino acids of: Arg551, Gln553, Tyr555, Thr558,and/or Arg560 (numbering based on the human sequence) and, e.g., doesnot bind pre-plasma kallikrein.

In some embodiments, the plasma kallikrein binding protein binds one ormore amino acids of: S478, N481, S525, and K526 (numbering based on thehuman kallikrein sequence). In other embodiments, the plasma kallikreinbinding protein binds two, three or four (i.e., all) amino acids of:S478, N481, S525, and K526 (numbering based on the human kallikreinsequence).

In some embodiments, the plasma kallikrein binding protein decreasesFactor XIIa and/or bradykinin production by greater than about 5%, about10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%,about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about75%, about 80%, about 85%, about 90%, or about 95% as compared to astandard, e.g., the Factor XIIa and/or bradykinin production under thesame conditions but in the absence of the protein.

In some embodiments, the plasma kallikrein binding protein has anapparent inhibition constant (K_(i,app)) of less than 1000, 500, 100,10, 1, 0.5 or 0.2 nM.

In one embodiment, the HC and LC variable domain sequences arecomponents of the same polypeptide chain.

In another embodiment, the HC and LC variable domain sequences arecomponents of different polypeptide chains. For example, the plasmakallikrein binding protein is an IgG., e.g., IgG1, IgG2, IgG3, or IgG4.The plasma kallikrein binding protein can be a soluble Fab (sFab).

In some embodiments, the plasma kallikrein binding protein has a serumresidence time of 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks or more, invivo, e.g., in humans. In one embodiment, the plasma kallikrein bindingprotein is an IgG, e.g., an IgG1, IgG2, IgG3 or IgG4, that has a serumresidence time of 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks or more invivo, e.g., in humans.

In some embodiments, the plasma kallikrein binding protein is physicallyassociated with a moiety that improves serum residence time, e.g., amoiety described herein.

In other embodiments, the plasma kallikrein binding protein includes aFab2′, scFv, minibody, scFv::Fc fusion, Fab::HSA fusion, HSA::Fabfusion, Fab::HSA::Fab fusion, or other molecule that comprises theantigen combining site of one of the binding proteins herein. The VH andVL regions of these Fabs can be provided as IgG, Fab, Fab2, Fab2′, scFv,PEGylated Fab, PEGylated scFv, PEGylated Fab2, VH::CH1::HSA+LC,HSA::VH::CH1+LC, LC::HSA+VH::CH1, HSA::LC+VH::CH1, or other appropriateconstruction.

In one embodiment, the plasma kallikrein binding protein is a human orhumanized antibody or is non-immunogenic in a human. For example, theprotein includes one or more human antibody framework regions, e.g., allhuman framework regions.

In one embodiment, the plasma kallikrein binding protein includes ahuman Fc domain, or an Fc domain that is at least 95, 96, 97, 98, or 99%identical to a human Fc domain.

In one embodiment, the plasma kallikrein binding protein is a primate orprimatized antibody or is non-immunogenic in a human. For example, theprotein includes one or more primate antibody framework regions, e.g.,all primate framework regions.

In one embodiment, the plasma kallikrein binding protein includes aprimate Fc domain, or an Fc domain that is at least 95, 96, 97, 98, or99% identical to a primate Fc domain. “Primate” includes humans (Homosapiens), chimpanzees (Pan troglodytes and Pan paniscus (bonobos)),gorillas (Gorilla gorilla), gibons, monkeys, lemurs, aye-ayes(Daubentonia madagascariensis), and tarsiers.

In one embodiment, the plasma kallikrein binding protein includes humanframework regions, or framework regions that are at least 95, 96, 97,98, or 99% identical to human framework regions.

In certain embodiments, the plasma kallikrein binding protein includesno sequences from mice or rabbits (e.g., is not a murine or rabbitantibody).

In certain embodiments, the plasma kallikrein binding protein is capableof binding to a cell or tissue, e.g., that expresses plasma kallikrein.

In one embodiment, the plasma kallikrein binding protein is physicallyassociated with a nanoparticle, and can be used to guide a nanoparticleto a cell or tissue expressing plasma kallikrein.

In some aspects, the disclosure features an isolated protein (e.g.,antibody, e.g., human antibody) that binds the same epitope or competesfor binding with a kallikrein binding protein described herein.

In some embodiments, the protein binds the same epitope or competes forbinding with a protein (e.g., epi-Kal2) and/or a small molecule (e.g.,AEBSF) described herein.

In some embodiments, the isolated protein comprises a heavy chainimmunoglobulin variable domain sequence and a light chain immunoglobulinvariable domain sequence, wherein:

the heavy chain immunoglobulin variable domain sequence comprises one,two, or three (e.g., three) CDR regions from the heavy chain variabledomain of a protein described herein, and/or

the light chain immunoglobulin variable domain sequence comprises one,two, or three (e.g., three) CDR regions from the light chain variabledomain of a protein described herein,

wherein the protein binds to plasma kallikrein.

In some embodiments, the heavy chain immunoglobulin variable domainsequence comprises one, two, or three (e.g., three) CDR regions from theheavy chain variable domain of M162-A04, M160-G12, M142-H08, X63-G06,X101-A01, X81-B01, X67-D03, X67-G04, X115-B07, X115-D05, X115-E09,X115-H06, X115-A03, X115-D01, X115-F02, X115-G04, M29-D09, M145-D11,M06-D09 and M35-G04.

the light chain immunoglobulin variable domain sequence comprises one,two, or three (e.g., three) CDR regions from the light chain variabledomain of M162-A04, M160-G12, M142-H08, X63-G06, X101-A01, X81-B01,X67-D03, X67-G04, X115-B07, X115-D05, X115-E09, X115-H06, X115-A03,X115-D01, X115-F02, X115-G04, M29-D09, M145-D11, M06-D09 and M35-G04(respectively).

In some embodiments, the protein inhibits plasma kallikrein (e.g., humanplasma kallikrein and/or mouse plasma kallikrein).

In some embodiments, the one, two, or three (e.g., three) CDR regionsfrom the heavy chain variable domain are from X81-B01 and/or the one,two, or three (e.g., three) CDR regions from the light chain variabledomain are from X81-B01.

In some embodiments, the one, two, or three (e.g., three) CDR regionsfrom the heavy chain variable domain are from X67-D03 and/or the one,two, or three (e.g., three) CDR regions from the light chain variabledomain are from X67-D03.

In some embodiments, the one, two, or three (e.g., three) CDR regionsfrom the heavy chain variable domain are from X63-G06 and/or the one,two, or three (e.g., three) CDR regions from the light chain variabledomain are from X63-G06.

In some embodiments, the one, two, or three (e.g., three) CDR regionsfrom the heavy chain variable domain are from M162-A04 and/or the one,two, or three (e.g., three) CDR regions from the light chain variabledomain are from MJ162-A04.

In some embodiments, the heavy chain immunoglobulin variable domainsequence comprises the heavy chain variable domain of a proteindescribed herein, and/or the light chain immunoglobulin variable domainsequence comprises the light chain variable domain of a proteindescribed herein.

In some embodiments, the heavy chain immunoglobulin variable domainsequence comprises the heavy chain variable domain of M162-A04,M160-G12, M142-H08, X63-G06, X101-A01, X81-B01, X67-D03, X67-G04,X115-B07, X115-D05, X115-E09, X115-H06, X115-A03, X115-D01, X115-F02,X115-G04, M29-D09, M145-D11, M06-D09 and M35-G04, and/or the light chainimmunoglobulin variable domain sequence comprises the light chainvariable domain of M162-A04, M160-G12, M142-H08, X63-G06, X101-A01,X81-B01, X67-D03, X67-G04, X115-B07, X115-D05, X115-E09, X115-H06,X115-A03, X115-D01, X115-F02, X115-G04, M29-D09, M145-D11, M06-D09 andM35-G04 (respectively).

In some embodiments, the heavy chain immunoglobulin variable domainsequence comprises the heavy chain variable domain of X81-B01, and/orthe light chain immunoglobulin variable domain sequence comprises thelight chain variable domain of X81-B01.

In some embodiments, the heavy chain immunoglobulin variable domainsequence comprises the heavy chain variable domain of X67-D03, and/orthe light chain immunoglobulin variable domain sequence comprises thelight chain variable domain of X67-D03.

In some embodiments, the protein comprises the heavy chain of a proteindescribed herein, and/or the light chain of a protein described herein.

In some embodiments, the protein comprises the heavy chain of M162-A04,M160-G12, M142-H08, X63-G06, X101-A01, X81-B01, X67-D03, X67-G04,X115-B07, X115-D05, X115-E09, X115-H06, X115-A03, X115-D01, X115-F02,X115-G04, M29-D09, M145-D11, M06-D09 and M35-G04.

and/or the light chain of M162-A04, M160-G12, M142-H08, X63-G06,X101-A01, X81-B01, X67-D03, X67-G04, X115-B07, X115-D05, X115-E09,X115-H06, X115-A03, X115-D01, X115-F02, X115-G04, M29-D09, M145-D11,M06-D09 and M35-G04 (respectively).

In some embodiments, the protein comprises the heavy chain of X81-B01,and/or the light chain of X81-B01.

In some embodiments, the protein comprises the heavy chain of X67-D03,and/or the light chain of X67-D03.

In some embodiments, the plasma kallikrein binding protein does not bindprekallikrein (e.g., human prekallikrein and/or mouse prekallikrein),but binds to the active form of plasma kallikrein (e.g., human plasmakallikrein and/or mouse plasma kallikrein).

In some embodiments, the plasma kallikrein binding protein decreasesFactor XIIa and/or bradykinin production by greater than about 5%, about10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%,about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about75%, about 80%, about 85%, about 90%, or about 95% as compared to astandard, e.g., the Factor XIIa and/or bradykinin production under thesame conditions but in the absence of the protein.

In some embodiments, the protein includes one or more of the followingcharacteristics: (a) a human CDR or human framework region; (b) the HCimmunoglobulin variable domain sequence comprises one or more (e.g., 1,2, or 3) CDRs that are at least 85, 88, 89, 90, 91, 92, 93, 94, 95, 96,97, 98, 99, or 100% identical to a CDR of a HC variable domain describedherein; (c) the LC immunoglobulin variable domain sequence comprises oneor more (e.g., 1, 2, or 3) CDRs that are at least 85, 88, 89, 90, 91,92, 93, 94, 95, 96, 97, 98, 99, or 100% identical to a CDR of a LCvariable domain described herein; (d) the LC immunoglobulin variabledomain sequence is at least 85, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97,98, 99, or 100% identical to a LC variable domain described herein(e.g., overall or in framework regions or CDRs); (e) the HCimmunoglobulin variable domain sequence is at least 85, 88, 89, 90, 91,92, 93, 94, 95, 96, 97, 98, 99, or 100% identical to a HC variabledomain described herein (e.g., overall or in framework regions or CDRs);(f) the protein binds an epitope bound by a protein described herein, orcompetes for binding with a protein described herein; (g) a primate CDRor primate framework region; (h) the HC immunoglobulin variable domainsequence comprises a CDR1 that differs by at least one amino acid but byno more than 2 or 3 amino acids from the CDR1 of a HC variable domaindescribed herein; (i) the HC immunoglobulin variable domain sequencecomprises a CDR2 that differs by at least one amino acid but by no morethan 2, 3, 4, 5, 6, 7, or 8 amino acids from the CDR2 of a HC variabledomain described herein; (j) the HC immunoglobulin variable domainsequence comprises a CDR3 that differs by at least one amino acid but byno more than 2, 3, 4, 5, or 6 amino acids from the CDR3 of a HC variabledomain described herein; (k) the LC immunoglobulin variable domainsequence comprises a CDR1 that differs by at least one amino acid but byno more than 2, 3, 4, or 5 amino acids from the CDR1 of a LC variabledomain described herein; (1) the LC immunoglobulin variable domainsequence comprises a CDR2 that differs by at least one amino acid but byno more than 2, 3, or 4 amino acids from the CDR2 of a LC variabledomain described herein; (m) the LC immunoglobulin variable domainsequence comprises a CDR3 that differs by at least one amino acid but byno more than 2, 3, 4, or 5 amino acids from the CDR3 of a LC variabledomain described herein; (n) the LC immunoglobulin variable domainsequence differs by at least one amino acid but by no more than 2, 3, 4,5, 6, 7, 8, 9, or 10 amino acids from a LC variable domain describedherein (e.g., overall or in framework regions or CDRs); and (o) the HCimmunoglobulin variable domain sequence differs by at least one aminoacid but by no more than 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids froma HC variable domain described herein (e.g., overall or in frameworkregions or CDRs).

In some embodiments, the protein has an apparent inhibition constant(K_(i,app)) of less than 1000, 500, 100, 10, 1, 0.5 or 0.2 nM.

In some embodiments, the antibody does not bind prekallikrein (e.g.,human prekallikrein and/or mouse prekallikrein), but binds to the activeform of plasma kallikrein (e.g., human plasma kallikrein and/or mouseplasma kallikrein).

In a preferred embodiment, the protein is an antibody (e.g., a humanantibody) having the light and heavy chains of antibodies selected fromthe group consisting of M162-A04, M160-G12, M142-H08, X63-G06, X101-A01,X81-B01, X67-D03, X67-G04, X115-B07, X115-D05, X115-E09, X115-H06,X115-A03, X115-D01, X115-F02, X115-G04, M29-D09, M145-D11, M06-D09 andM35-G04.

In a preferred embodiment, the protein is an antibody (e.g., a humanantibody) having the heavy chain of an antibody selected from the groupconsisting of: M162-A04, M160-G12, M142-H08, X63-G06, X101-A01, X81-B01,X67-D03, X67-G04, X115-B07, X115-D05, X115-E09, X115-H06, X115-A03,X115-D01, X115-F02, X115-G04, M29-D09, M145-D11, M06-D09 and M35-G04.

In a preferred embodiment, the protein is an antibody (e.g., a humanantibody) having the light chain of an antibody selected from the groupconsisting of: M162-A04, M160-G12, M142-H08, X63-G06, X101-A01, X81-B01,X67-D03, X67-G04, X115-B07, X115-D05, X115-E09, X115-H06, X115-A03,X115-D01, X115-F02, X115-G04, M29-D09, M145-D11, M06-D09 and M35-G04.

In a preferred embodiment, the protein is an antibody (e.g., a humanantibody) having light and heavy antibody variable regions of anantibody selected from the group consisting of M162-A04, M160-G12,M142-H08, X63-G06, X101-A01, X81-B01, X67-D03, X67-G04, X115-B07,X115-D05, X115-E09, X115-H06, X115-A03, X115-D01, X115-F02, X115-G04,M29-D09, M145-D11, M06-D09 and M35-G04.

In a preferred embodiment, the protein is an antibody (e.g., a humanantibody) having a heavy chain antibody variable region of an antibodyselected from the group consisting of: M162-A04, M160-G12, M142-H08,X63-G06, X101-A01, X81-B01, X67-D03, X67-G04, X115-B07, X115-D05,X115-E09, X115-H06, X115-A03, X115-D01, X115-F02, X115-G04, M29-D09,M145-D11, M06-D09 and M35-G04.

In a preferred embodiment, the protein is an antibody (e.g., a humanantibody) having a light chain antibody variable region of an antibodyselected from the group consisting of: M162-A04, M160-G12, M142-H08,X63-G06, X101-A01, X81-B01, X67-D03, X67-G04, X115-B07, X115-D05,X115-E09, X115-H06, X115-A03, X115-D01, X115-F02, X115-G04, M29-D09,M145-D11, M06-D09 and M35-G04.

In a preferred embodiment, the protein is an antibody (e.g., a humanantibody) having one or more (e.g., 1, 2, or 3) heavy chain CDRsselected from the corresponding CDRs of the group of heavy chainsconsisting of M162-A04, M160-G12, M142-H08, X63-G06, X101-A01, X81-B01,X67-D03, X67-G04, X115-B07, X115-D05, X115-E09, X115-H06, X115-A03,X115-D01, X115-F02, X115-G04, M29-D09, M145-D11, M06-D09 and M35-G04.

In a preferred embodiment, the protein is an antibody (e.g., a humanantibody) having one or more (e.g., 1, 2, or 3) light chain CDRsselected from the corresponding CDRs of the group of light chainsconsisting of M162-A04, M160-G12, M142-H08, X63-G06, X101-A01, X81-B01,X67-D03, X67-G04, X115-B07, X115-D05, X115-E09, X115-H06, X115-A03,X115-D01, X115-F02, X115-G04, M29-D09, M145-D11, M06-D09 and M35-G04.

In a preferred embodiment, the protein is an antibody (e.g., a humanantibody) having one or more (e.g., 1, 2, or 3) heavy chain CDRsselected from the corresponding CDRs of the group of heavy chainsconsisting of M162-A04, M160-G12, M142-H08, X63-G06, X101-A01, X81-B01,X67-D03, X67-G04, X115-B07, X115-D05, X115-E09, X115-H06, X115-A03,X115-D01, X115-F02, X115-G04, M29-D09, M145-D11, M06-D09 and M35-G04.

and one or more (e.g., 1, 2, or 3) light chain CDRs selected from thecorresponding CDRs of the group of light chains consisting of M162-A04,M160-G12, M142-H08, X63-G06, X101-A01, X81-B01, X67-D03, X67-G04,X115-B07, X115-D05, X115-E09, X115-H06, X115-A03, X115-D01, X115-F02,X115-G04, M29-D09, M145-D11, M06-D09 and M35-G04 (respectively).

In a preferred embodiment, the protein is an antibody (e.g., a humanantibody) having the light and heavy chains of X81-B01.

In a preferred embodiment, the protein is an antibody (e.g., a humanantibody) having the heavy chain of X81-B01.

In a preferred embodiment, the protein is an antibody (e.g., a humanantibody) having the light chain of X81-B01.

In a preferred embodiment, the protein is an antibody (e.g., a humanantibody) having light and heavy antibody variable regions of anantibody selected from X81-B01.

In a preferred embodiment, the protein is an antibody (e.g., a humanantibody) having a heavy chain antibody variable region of X81-B01.

In a preferred embodiment, the protein is an antibody (e.g., a humanantibody) having a light chain antibody variable region of X81-B01.

In a preferred embodiment, the protein is an antibody (e.g., a humanantibody) having one or more (e.g., 1, 2, or 3) heavy chain CDRs fromthe corresponding CDRs of the heavy chain of X81-B01.

In a preferred embodiment, the protein is an antibody (e.g., a humanantibody) having one or more (e.g., 1, 2, or 3) light chain CDRs fromthe corresponding CDRs of the light chain of X81-B01.

In a preferred embodiment, the protein is an antibody (e.g., a humanantibody) having one or more (e.g., 1, 2, or 3) heavy chain CDRs fromthe heavy chain of X81-B01 and one or more (e.g., 1, 2, or 3) lightchain CDRs from the corresponding CDRs of the light chain of X81-B01.

In a preferred embodiment, the protein is an antibody (e.g., a humanantibody) having the light and heavy chains of X67-D03.

In a preferred embodiment, the protein is an antibody (e.g., a humanantibody) having the heavy chain of X67-D03.

In a preferred embodiment, the protein is an antibody (e.g., a humanantibody) having the light chain of X67-D03.

In a preferred embodiment, the protein is an antibody (e.g., a humanantibody) having light and heavy antibody variable regions of anantibody selected from X67-D03.

In a preferred embodiment, the protein is an antibody (e.g., a humanantibody) having a heavy chain antibody variable region of X67-D03.

In a preferred embodiment, the protein is an antibody (e.g., a humanantibody) having a light chain antibody variable region of X67-D03.

In a preferred embodiment, the protein is an antibody (e.g., a humanantibody) having one or more (e.g., 1, 2, or 3) heavy chain CDRs fromthe corresponding CDRs of the heavy chain of X67-D03.

In a preferred embodiment, the protein is an antibody (e.g., a humanantibody) having one or more (e.g., 1, 2, or 3) light chain CDRs fromthe corresponding CDRs of the light chain of X67-D03.

In a preferred embodiment, the protein is an antibody (e.g., a humanantibody) having one or more (e.g., 1, 2, or 3) heavy chain CDRs fromthe heavy chain of X67-D03 and one or more (e.g., 1, 2, or 3) lightchain CDRs from the corresponding CDRs of the light chain of X67-D03.

In one embodiment, the HC and LC variable domain sequences arecomponents of the same polypeptide chain.

In some embodiments, the plasma kallikrein binding protein has a serumresidence time of 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks or more, invivo, e.g., in humans. In one embodiment, the plasma kallikrein bindingprotein is an IgG, e.g., an IgG1, IgG2, IgG3 or IgG4, that has a serumresidence time of 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks or more invivo, e.g., in humans.

In some embodiments, the plasma kallikrein binding protein is physicallyassociated with a moiety that improves serum residence time, e.g., amoiety described herein.

In another embodiment, the HC and LC variable domain sequences arecomponents of different polypeptide chains. For example, the protein isan IgG, e.g., IgG1, IgG2, IgG3, or IgG4. The protein can be a solubleFab (sFab).

In other embodiments, the protein includes a Fab2′, scFv, minibody,scFv::Fc fusion, Fab::HSA fusion, HSA::Fab fusion, Fab::HSA::Fab fusion,or other molecule that comprises the antigen combining site of one ofthe binding proteins herein. The VH and VL regions of these Fabs can beprovided as IgG, Fab, Fab2, Fab2′, scFv, PEGylated Fab, PEGylated scFv,PEGylated Fab2, VH::CH1::HSA+LC, HSA::VH::CH1+LC, LC::HSA+VH::CH1,HSA::LC+VH::CH1, or other appropriate construction.

In one embodiment, the protein is a human or humanized antibody or isnon-immunogenic in a human. For example, the protein includes one ormore human antibody framework regions, e.g., all human frameworkregions.

In one embodiment, the protein includes a human Fc domain, or an Fcdomain that is at least 95, 96, 97, 98, or 99% identical to a human Fcdomain.

In one embodiment, the protein is a primate or primatized antibody or isnon-immunogenic in a human. For example, the protein includes one ormore primate antibody framework regions, e.g., all primate frameworkregions.

In one embodiment, the protein includes a primate Fc domain, or an Fcdomain that is at least 95, 96, 97, 98, or 99% identical to a primate Fcdomain. “Primate” includes humans (Homo sapiens), chimpanzees (Pantroglodytes and Pan paniscus (bonobos)), gorillas (Gorilla gorilla),gibons, monkeys, lemurs, aye-ayes (Daubentonia madagascariensis), andtarsiers.

In one embodiment, the protein includes human framework regions, orframework regions that are at least 95, 96, 97, 98, or 99% identical tohuman framework regions.

In certain embodiments, the protein includes no sequences from mice orrabbits (e.g., is not a murine or rabbit antibody).

In certain embodiments, the protein is capable of binding to a cell ortissue, e.g., that expresses plasma kallikrein.

In one embodiment, protein is physically associated with a nanoparticle,and can be used to guide a nanoparticle to a cell or tissue expressingplasma kallikrein.

In some aspects, the disclosure features a pharmaceutical compositioncomprising a kallikrein binding protein described herein, e.g.,including a pharmaceutically acceptable carrier. In some embodiments,the composition can be at least 10, 20, 30, 50, 75, 85, 90, 95, 98, 99,or 99.9% free of other protein species. In one embodiment, thepharmaceutical composition can be at least 10, 20, 30, 50, 75, 85, 90,95, 98, 99, or 99.9% free of fragments of the binding protein that donot binding plasma kallikrein (e.g., human plasma kallikrein) or bindplasma kallikrein (e.g., human plasma kallikrein with a Ki, app of 5000nM or greater.

In some aspects, the disclosure features a method of treating orpreventing a plasma kallikrein associated disorder in a subject, themethod comprising:

administering an isolated protein (e.g., antibody, e.g., human antibody)that binds plasma kallikrein (e.g., human plasma kallikrein and/or mouseplasma kallikrein) and, e.g., does not bind prekallikrein (e.g., humanprekallikrein and/or mouse prekallikrein) to the subject,

In some embodiments, the protein binds the same epitope or competes forbinding with a protein (e.g., epi-Kal2) and/or a small molecule (e.g.,AEBSF) described herein.

In some embodiments, the protein binds the same epitope or competes forbinding with a kallikrein binding protein described herein.

In some embodiments, the plasma kallikrein associated disorder isselected from the group consisting of rheumatoid arthritis, gout,intestinal bowel disease, oral mucositis, neuropathic pain, inflammatorypain, spinal stenosis-degenerative spine disease, arterial or venousthrombosis, post operative ileus, aortic aneurysm, osteoarthritis,vasculitis, edema, hereditary angioedema, cerebral edema, pulmonaryembolism, stroke, clotting on ventricular assistance devices or stents,head trauma or peri-tumor brain edema, sepsis, acute middle cerebralartery (MCA) ischemic event (stroke), restenosis (e.g., afterangioplasty), systemic lupus erythematosis nephritis, and burn injury.In some embodiments, the plasma kallikrein binding protein reducesabberent clotting associated with the contact activation system (i.e.,intrinsic activation system) by at least 10% as measured by e.g., anAPTT clotting assay. In other embodiments, the plasma kallikrein bindingprotein reduces abberent clotting associated with the contact activationsystem by at least 20%, at least 30%, at least 40%, at least 50%, atleast 60%, at least 70%, at least 80%, at least 90%, at least 95%, atleast 99%, or even 100% (i.e., no detectable abberent clotting).

In some embodiments, the plasma kallikrein binding protein isadministered in combination with another treatment for the disorder.

In some embodiments, the protein described herein is selected from thegroup consisting of M162-A04, M160-G12, M142-H08, X63-G06, X101-A01,X81-B01, X67-D03, X67-G04, X115-B07, X115-D05, X115-E09, X115-H06,X115-A03, X115-D01, X115-F02, X115-G04, M29-D09, M145-D11, M06-D09 andM35-G04.

In some embodiments, the plasma kallikrein binding protein competes withor binds the same epitope as X81-B01.

In some embodiments, the plasma kallikrein binding protein competes withor binds the same epitope as X67-D03.

In some embodiments, the plasma kallikrein binding protein does not bindprekallikrein (e.g., human prekallikrein and/or mouse prekallikrein),but binds to the active form of plasma kallikrein (e.g., human plasmakallikrein and/or mouse plasma kallikrein).

In certain embodiments, the protein binds at or near the active site ofthe catalytic domain of plasma kallikrein, or a fragment thereof, orbinds an epitope that overlaps with the active site of plasmakallikrein.

In some embodiments, the protein binds to one or more amino acids thatform the catalytic triad of plasma kallikrein: His434, Asp483, and/orSer578 (numbering based on the human sequence).

In some embodiments, the protein binds to one or more amino acids ofSer479, Tyr563, and/or Asp585 (numbering based on the human sequence).

In other embodiments, the protein binds to one or more amino acids ofArg551, Gln553, Tyr555, Thr558, and/or Arg560 (numbering based on thehuman sequence). In some embodiments, the plasma kallikrein bindingprotein binds one or more amino acids of: S478, N481, S525, and K526(numbering based on the human kallikrein sequence).

In some embodiments, the plasma kallikrein binding protein decreasesFactor XIIa and/or bradykinin production by greater than about 5%, about10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%,about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about75%, about 80%, about 85%, about 90%, or about 95% as compared to astandard, e.g., the Factor XIIa and/or bradykinin production under thesame conditions but in the absence of the protein.

In some embodiments, the plasma kallikrein binding protein has anapparent inhibition constant (K_(i,app)) of less than 1000, 500, 100,10, 5, 1, 0.5, or 0.2 nM.

In one embodiment, the HC and LC variable domain sequences arecomponents of the same polypeptide chain.

In some embodiments, the plasma kallikrein binding protein has a serumresidence time of 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks or more, invivo, e.g., in humans. In one embodiment, the plasma kallikrein bindingprotein is an IgG, e.g., an IgG1, IgG2, IgG3 or IgG4, that has a serumresidence time of 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks or more invivo, e.g., in humans.

In some embodiments, the plasma kallikrein binding protein is physicallyassociated with a moiety that improves serum residence time, e.g., amoiety described herein.

In another embodiment, the HC and LC variable domain sequences arecomponents of different polypeptide chains. For example, the plasmakallikrein binding protein is an IgG, e.g., IgG1, IgG2, IgG3, or IgG4.The plasma kallikrein binding protein can be a soluble Fab (sFab).

In other implementations the plasma kallikrein binding protein includesa Fab2′, scFv, minibody, scFv::Fc fusion, Fab::HSA fusion, HSA::Fabfusion, Fab::HSA::Fab fusion, or other molecule that comprises theantigen combining site of one of the binding proteins herein. The VH andVL regions of these Fabs can be provided as IgG, Fab, Fab2, Fab2′, scFv,PEGylated Fab, PEGylated scFv, PEGylated Fab2, VH::CH1::HSA+LC,HSA::VH::CH1+LC, LC::HSA+VH::CH1, HSA::LC+VH::CH1, or other appropriateconstruction.

In one embodiment, the plasma kallikrein binding protein is a human orhumanized antibody or is non-immunogenic in a human. For example, theprotein includes one or more human antibody framework regions, e.g., allhuman framework regions.

In one embodiment, the plasma kallikrein binding protein includes ahuman Fc domain, or an Fc domain that is at least 95, 96, 97, 98, or 99%identical to a human Fc domain.

In one embodiment, the plasma kallikrein binding protein is a primate orprimatized antibody or is non-immunogenic in a human. For example, theprotein includes one or more primate antibody framework regions, e.g.,all primate framework regions.

In one embodiment, the plasma kallikrein binding protein includes aprimate Fc domain, or an Fc domain that is at least 95, 96, 97, 98, or99% identical to a primate Fc domain. “Primate” includes humans (Homosapiens), chimpanzees (Pan troglodytes and Pan paniscus (bonobos)),gorillas (Gorilla gorilla), gibons, monkeys, lemurs, aye-ayes(Daubentonia madagascariensis), and tarsiers.

In one embodiment, the plasma kallikrein binding protein includes humanframework regions, or framework regions that are at least 95, 96, 97,98, or 99% identical to human framework regions.

In certain embodiments, the plasma kallikrein binding protein includesno sequences from mice or rabbits (e.g., is not a murine or rabbitantibody).

In certain embodiments, the plasma kallikrein binding protein is capableof binding to a cell or tissue, e.g., that expresses plasma kallikrein.

In one embodiment, the plasma kallikrein binding protein is physicallyassociated with a nanoparticle, and can be used to guide a nanoparticleto a cell or tissue expressing plasma kallikrein.

A method of treating or preventing a plasma kallikrein associateddisorder in a subject, the method comprising:

administering an isolated protein (e.g., antibody, e.g., human antibody)comprising a heavy chain immunoglobulin variable domain sequence and alight chain immunoglobulin variable domain sequence to the subject,wherein:

the heavy chain immunoglobulin variable domain sequence comprises one,two, or three (e.g., three) CDR regions from the heavy chain variabledomain of a protein described herein, and/or

the light chain immunoglobulin variable domain sequence comprises one,two, or three (e.g., three) CDR regions from the light chain variabledomain of a protein described herein,

wherein the protein binds to plasma kallikrein (e.g., human plasmakallikrein and/or mouse plasma kallikrein).

In some embodiments, the plasma kallikrein associated disorder isselected from the group consisting of rheumatoid arthritis, gout,intestinal bowel disease, oral mucositis, neuropathic pain, inflammatorypain, spinal stenosis-degenerative spine disease, arterial or venousthrombosis, post operative ileus, aortic aneurysm, osteoarthritis,vasculitis, edema, hereditary angioedema, cerebral edema, pulmonaryembolism, stroke, clotting of ventrical assistance devices or stents,head trauma or peri-tumor brain edema, sepsis, acute middle cerebralartery (MCA) ischemic event (stroke), restenosis (e.g., afterangioplasty), systemic lupus erythematosis nephritis, and burn injury.In some embodiments, the plasma kallikrein binding protein reducesabberent clotting associated with the contact activation system (i.e.,intrinsic activation system) by at least 10% as measured by e.g., anAPTT clotting assay (e.g., by at least 20%, at least 30%, at least 40%,at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, atleast 95%, at least 99%, or even 100% (i.e., no detectable abberentclotting)).

In some embodiments, the protein is administered in combination withanother treatment for the disorder.

In some embodiments, the protein is administered in combination with asecond agent selected from the group consisting of ecallantide, a C1esterase inhibitor, aprotinin, a bradykinin B2 receptor inhibitor (e.g.,icatibant).

In some embodiments, the heavy chain immunoglobulin variable domainsequence comprises one, two, or three (e.g., three) CDR regions from theheavy chain variable domain of M162-A04, M160-G12, M142-H08, X63-G06,X101-A01, X81-B01, X67-D03, X67-G04, X115-B07, X115-D05, X115-E09,X115-H06, X115-A03, X115-D01, X115-F02, X115-G04, M29-D09, M145-D11,M06-D09 and M35-G04, and/or

the light chain immunoglobulin variable domain sequence comprises one,two, or three (e.g., three) CDR regions from the light chain variabledomain of M162-A04, M160-G12, M142-H08, X63-G06, X101-A01, X81-B01,X67-D03, X67-G04, X115-B07, X115-D05, X115-E09, X115-H06, X115-A03,X115-D01, X115-F02, X115-G04, M29-D09, M145-D11, M06-D09 and M35-G04(respectively).

In some embodiments, the protein inhibits plasma kallikrein.

In some embodiments, the one, two, or three (e.g., three) CDR regionsfrom the heavy chain variable domain are from X81-B01 and/or the one,two, or three (e.g., three) CDR regions from the light chain variabledomain are from X81-B01.

In some embodiments, the one, two, or three (e.g., three) CDR regionsfrom the heavy chain variable domain are from X67-D03 and/or the one,two, or three (e.g., three) CDR regions from the light chain variabledomain are from X67-D03.

In some embodiments, the heavy chain immunoglobulin variable domainsequence comprises the heavy chain variable domain of a proteindescribed herein, and/or the light chain immunoglobulin variable domainsequence comprises the light chain variable domain of a proteindescribed herein.

In some embodiments, the heavy chain immunoglobulin variable domainsequence comprises the heavy chain variable domain of M162-A04,M160-G12, M142-H08 X63-G06, X101-A01, X81-B01, X67-D03, X67-G04,X115-B07, X115-D05, X115-E09, X115-H06, X115-A03, X115-D01, X115-F02,X115-G04, M29-D09, M145-D11, M06-D09 and M35-G04, and/or the light chainimmunoglobulin variable domain sequence comprises the light chainvariable domain of M162-A04, M160-G12, M142-H08, X63-G06, X101-A01,X81-B01, X67-D03, X67-G04, X115-B07, X115-D05, X115-E09, X115-H06,X115-A03, X115-D01, X115-F02, X115-G04, M29-D09, M145-D11, M06-D09 andM35-G04.

In some embodiments, the heavy chain immunoglobulin variable domainsequence comprises the heavy chain variable domain of X81-B01, and/orthe light chain immunoglobulin variable domain sequence comprises thelight chain variable domain of X81-B01.

In some embodiments, the heavy chain immunoglobulin variable domainsequence comprises the heavy chain variable domain of X67-D03, and/orthe light chain immunoglobulin variable domain sequence comprises thelight chain variable domain of X67-D03.

In some embodiments, the protein comprises the heavy chain of a proteindescribed herein, and/or the light chain of a protein described herein.

In some embodiments, the protein comprises the heavy chain of M162-A04,M160-G12, M142-H08, X63-G06, X101-A01, X81-B01, X67-D03, X67-G04,X115-B07, X115-D05, X115-E09, X115-H06, X115-A03, X115-D01, X115-F02,X115-G04, M29-D09, M145-D11, M06-D09 and M35-G04, and/or the light chainof M162-A04, M160-G12, M142-H08, X63-G06, X101-A01, X81-B01, X67-D03,X67-G04, X115-B07, X115-D05, X115-E09, X115-H06, X115-A03, X115-D01,X115-F02, X115-G04, M29-D09, M145-D11, M06-D09 and M35-G04(respectively).

In some embodiments, the protein comprises the heavy chain of X81-B01,and/or the light chain of X81-B01.

In some embodiments, the protein comprises the heavy chain of X67-D03,and/or the light chain of X67-D03.

In some embodiments, the plasma kallikrein binding protein does not bindprekallikrein (e.g., human prekallikrein and/or murine prekallikrein),but binds to the active form of plasma kallikrein (e.g., human plasmakallikrein and/or murine plasma kallikrein).

In some embodiments, the plasma kallikrein binding protein decreasesFactor XIIa and/or bradykinin production by greater than about 5%, about10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%,about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about75%, about 80%, about 85%, about 90%, or about 95% as compared to astandard, e.g., the Factor XIIa and/or bradykinin production under thesame conditions but in the absence of the protein.

In some embodiments, the protein includes one or more of the followingcharacteristics: (a) a human CDR or human framework region; (b) the HCimmunoglobulin variable domain sequence comprises one or more (e.g., 1,2, or 3) CDRs that are at least 85, 88, 89, 90, 91, 92, 93, 94, 95, 96,97, 98, 99, or 100% identical to a CDR of a HC variable domain describedherein; (c) the LC immunoglobulin variable domain sequence comprises oneor more (e.g., 1, 2, or 3) CDRs that are at least 85, 88, 89, 90, 91,92, 93, 94, 95, 96, 97, 98, 99, or 100% identical to a CDR of a LCvariable domain described herein; (d) the LC immunoglobulin variabledomain sequence is at least 85, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97,98, 99, or 100% identical to a LC variable domain described herein(e.g., overall or in framework regions or CDRs); (e) the HCimmunoglobulin variable domain sequence is at least 85, 88, 89, 90, 91,92, 93, 94, 95, 96, 97, 98, 99, or 100% identical to a HC variabledomain described herein (e.g., overall or in framework regions or CDRs);(f) the protein binds an epitope bound by a protein described herein, orcompetes for binding with a protein described herein; and (g) a primateCDR or primate framework region.

In some embodiments, the protein has an apparent inhibition constant(K_(i,app)) of less than 1000, 500, 100, 10, 5, 1, 0.5 or 0.2 nM.

In some embodiments, the antibody does not bind prekallikrein (e.g.,human prekallikrein), but binds to the active form of plasma kallikrein(e.g., human plasma kallikrein).

In a preferred embodiment, the protein is an antibody (e.g., a humanantibody) having the light and heavy chains of antibodies selected fromthe group consisting of M162-A04, M160-G12, M142-H08, X63-G06, X101-A01,X81-B01, X67-D03, X67-G04, X115-B07, X115-D05, X115-E09, X115-H06,X115-A03, X115-D01, X115-F02, X115-G04, M29-D09, M145-D11, M06-D09 andM35-G04.

In a preferred embodiment, the protein is an antibody (e.g., a humanantibody) having the heavy chain of an antibody selected from the groupconsisting of: M162-A04, M160-G12, M142-H08, X63-G06, X101-A01, X81-B01,X67-D03, X67-G04, X115-B07, X115-D05, X115-E09, X115-H06, X115-A03,X115-D01, X115-F02, X115-G04, M29-D09, M145-D11, M06-D09 and M35-G04.

In a preferred embodiment, the protein is an antibody (e.g., a humanantibody) having the light chain of an antibody selected from the groupconsisting of: M162-A04, M160-G12, M142-H08, X63-G06, X101-A01, X81-B01,X67-D03, X67-G04, X115-B07, X115-D05, X115-E09, X115-H06, X115-A03,X115-D01, X115-F02, X115-G04, M29-D09, M145-D11, M06-D09 and M35-G04.

In a preferred embodiment, the protein is an antibody (e.g., a humanantibody) having light and heavy antibody variable regions of anantibody selected from the group consisting of M162-A04, M160-G12,M142-H08, X63-G06, X101-A01, X81-B01, X67-D03, X67-G04, X115-B07,X115-D05, X115-E09, X115-H06, X115-A03, X115-D01, X115-F02, X115-G04,M29-D09, M145-D11, M06-D09 and M35-G04.

In a preferred embodiment, the protein is an antibody (e.g., a humanantibody) having a heavy chain antibody variable region of an antibodyselected from the group consisting of: M162-A04, M160-G12, M142-H08,X63-G06, X101-A01, X81-B01, X67-D03, X67-G04, X115-B07, X115-D05,X115-E09, X115-H06, X115-A03, X115-D01, X115-F02, X115-G04, M29-D09,M145-D11, M06-D09 and M35-G04.

In a preferred embodiment, the protein is an antibody (e.g., a humanantibody) having a light chain antibody variable region of an antibodyselected from the group consisting of: M162-A04, M160-G12, M142-H08,X63-G06, X101-A01, X81-B01, X67-D03, X67-G04, X115-B07, X115-D05,X115-E09, X115-H06, X115-A03, X115-D01, X115-F02, X115-G04, M29-D09,M145-D11, M06-D09 and M35-G04.

In a preferred embodiment, the protein is an antibody (e.g., a humanantibody) having one or more (e.g., 1, 2, or 3) heavy chain CDRsselected from the corresponding CDRs of the group of heavy chainsconsisting of M162-A04, M160-G12, M142-H08, X63-G06, X101-A01, X81-B01,X67-D03, X67-G04, X115-B07, X115-D05, X115-E09, X115-H06, X115-A03,X115-D01, X115-F02, X115-G04, M29-D09, M145-D11, M06-D09 and M35-G04.

In a preferred embodiment, the protein is an antibody (e.g., a humanantibody) having one or more (e.g., 1, 2, or 3) light chain CDRsselected from the corresponding CDRs of the group of light chainsconsisting of M162-A04, M160-G12, M142-H08, X63-G06, X101-A01, X81-B01,X67-D03, X67-G04, X115-B07, X115-D05, X115-E09, X115-H06, X115-A03,X115-D01, X115-F02, X115-G04, M29-D09, M145-D11, M06-D09 and M35-G04.

In a preferred embodiment, the protein is an antibody (e.g., a humanantibody) having one or more (e.g., 1, 2, or 3) heavy chain CDRsselected from the corresponding CDRs of the group of heavy chainsconsisting of M162-A04, M160-G12, M142-H08, X63-G06, X101-A01, X81-B01,X67-D03, X67-G04, X115-B07, X115-D05, X115-E09, X115-H06, X115-A03,X115-D01, X115-F02, X115-G04, M29-D09, M145-D11, M06-D09 and M35-G04 andone or more (e.g., 1, 2, or 3) light chain CDRs selected from thecorresponding CDRs of the group of light chains consisting of M162-A04,M160-G12, M142-H08, X63-G06, X101-A01, X81-B01, X67-D03, X67-G04,X115-B07, X115-D05, X115-E09, X115-H06, X115-A03, X115-D01, X115-F02,X115-G04, M29-D09, M145-D11, M06-D09 and M35-G04 (respectively).

In a preferred embodiment, the protein is an antibody (e.g., a humanantibody) having the light and heavy chains of X81-B01.

In a preferred embodiment, the protein is an antibody (e.g., a humanantibody) having the heavy chain of X81-B01.

In a preferred embodiment, the protein is an antibody (e.g., a humanantibody) having the light chain of X81-B01.

In a preferred embodiment, the protein is an antibody (e.g., a humanantibody) having light and heavy antibody variable regions of anantibody selected from X81-B01.

In a preferred embodiment, the protein is an antibody (e.g., a humanantibody) having a heavy chain antibody variable region of X81-B01.

In a preferred embodiment, the protein is an antibody (e.g., a humanantibody) having a light chain antibody variable region of X81-B01.

In a preferred embodiment, the protein is an antibody (e.g., a humanantibody) having one or more (e.g., 1, 2, or 3) heavy chain CDRs fromthe corresponding CDRs of the heavy chain of X81-B01.

In a preferred embodiment, the protein is an antibody (e.g., a humanantibody) having one or more (e.g., 1, 2, or 3) light chain CDRs fromthe corresponding CDRs of the light chain of X81-B01.

In a preferred embodiment, the protein is an antibody (e.g., a humanantibody) having one or more (e.g., 1, 2, or 3) heavy chain CDRs fromthe heavy chain of X81-B01 and one or more (e.g., 1, 2, or 3) lightchain CDRs from the corresponding CDRs of the light chain of X81-B01.

In a preferred embodiment, the protein is an antibody (e.g., a humanantibody) having the light and heavy chains of X67-D03.

In a preferred embodiment, the protein is an antibody (e.g., a humanantibody) having the heavy chain of X67-D03.

In a preferred embodiment, the protein is an antibody (e.g., a humanantibody) having the light chain of X67-D03.

In a preferred embodiment, the protein is an antibody (e.g., a humanantibody) having light and heavy antibody variable regions of anantibody selected from X67-D03.

In a preferred embodiment, the protein is an antibody (e.g., a humanantibody) having a heavy chain antibody variable region of X67-D03.

In a preferred embodiment, the protein is an antibody (e.g., a humanantibody) having a light chain antibody variable region of X67-D03.

In a preferred embodiment, the protein is an antibody (e.g., a humanantibody) having one or more (e.g., 1, 2, or 3) heavy chain CDRs fromthe corresponding CDRs of the heavy chain of X67-D03.

In a preferred embodiment, the protein is an antibody (e.g., a humanantibody) having one or more (e.g., 1, 2, or 3) light chain CDRs fromthe corresponding CDRs of the light chain of X67-D03.

In a preferred embodiment, the protein is an antibody (e.g., a humanantibody) having one or more (e.g., 1, 2, or 3) heavy chain CDRs fromthe heavy chain of X67-D03 and one or more (e.g., 1, 2, or 3) lightchain CDRs from the corresponding CDRs of the light chain of X67-D03.

In one embodiment, the HC and LC variable domain sequences arecomponents of the same polypeptide chain.

In some embodiments, the plasma kallikrein binding protein has a serumresidence time of 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks or more, invivo, e.g., in humans. In one embodiment, the plasma kallikrein bindingprotein is an IgG, e.g., an IgG1, IgG2, IgG3 or IgG4, that has a serumresidence time of 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks or more invivo, e.g., in humans.

In some embodiments, the plasma kallikrein binding protein is physicallyassociated with a moiety that improves serum residence time, e.g., amoiety described herein.

In another embodiment, the HC and LC variable domain sequences arecomponents of different polypeptide chains. For example, the protein isan IgG, e.g., IgG1, IgG2, IgG3, or IgG4. The protein can be a solubleFab (sFab).

In other implementations the protein includes a Fab2′, scFv, minibody,scFv::Fc fusion, Fab::HSA fusion, HSA::Fab fusion, Fab::HSA::Fab fusion,or other molecule that comprises the antigen combining site of one ofthe binding proteins herein. The VH and VL regions of these Fabs can beprovided as IgG, Fab, Fab2, Fab2′, scFv, PEGylated Fab, PEGylated scFv,PEGylated Fab2, VH::CH1::HSA+LC, HSA::VH::CH1+LC, LC::HSA+VH::CH1,HSA::LC+VH::CH1, or other appropriate construction.

In one embodiment, the protein is a human or humanized antibody or isnon-immunogenic in a human. For example, the protein includes one ormore human antibody framework regions, e.g., all human frameworkregions.

In one embodiment, the protein includes a human Fc domain, or an Fcdomain that is at least 95, 96, 97, 98, or 99% identical to a human Fcdomain.

In one embodiment, the protein is a primate or primatized antibody or isnon-immunogenic in a human. For example, the protein includes one ormore primate antibody framework regions, e.g., all primate frameworkregions.

In one embodiment, the protein includes a primate Fc domain, or an Fcdomain that is at least 95, 96, 97, 98, or 99% identical to a primate Fcdomain. “Primate” includes humans (Homo sapiens), chimpanzees (Pantroglodytes and Pan paniscus (bonobos)), gorillas (Gorilla gorilla),gibons, monkeys, lemurs, aye-ayes (Daubentonia madagascariensis), andtarsiers.

In one embodiment, the protein includes human framework regions, orframework regions that are at least 95, 96, 97, 98, or 99% identical tohuman framework regions.

In certain embodiments, the protein includes no sequences from mice orrabbits (e.g., is not a murine or rabbit antibody).

In certain embodiments, the protein is capable of binding to a cell ortissue, e.g., that expresses plasma kallikrein.

In one embodiment, protein is physically associated with a nanoparticle,and can be used to guide a nanoparticle to a cell or tissue expressingplasma kallikrein.

In some aspects, the disclosure features a method of promoting woundhealing in a subject, the method comprising:

administering an isolated protein (e.g., antibody, e.g., human antibody)that binds plasma kallikrein (e.g., human plasma kallikrein and/or mouseplasma kallikrein) and, e.g., does not bind prekallikrein (e.g., humanprekallikrein and/or mouse prekallikrein) to the subject.

In some embodiments, the protein binds the same epitope or competes forbinding with a kallikrein binding protein described herein. In someembodiments, the protein binds the same epitope or competes for bindingwith a protein (e.g., epi-Kal2) and/or a small molecule (e.g., AEBSF)described herein.

In some embodiments, the plasma kallikrein binding protein isadministered in combination with another treatment for wound healing.

In some embodiments, the protein described herein is selected from thegroup consisting of M162-A04, M160-G12, M142-H08, X63-G06, X101-A01,X81-B01, X67-D03, X67-G04, X115-B07, X115-D05, X115-E09, X115-H06,X115-A03, X115-D01, X115-F02, X115-G04, M29-D09, M145-D11, M06-D09 andM35-G04.

In some embodiments, the plasma kallikrein binding protein competes withor binds the same epitope as X81-B01.

In some embodiments, the plasma kallikrein binding protein competes withor binds the same epitope as X67-D03.

In some embodiments, the plasma kallikrein binding protein does not bindprekallikrein (e.g., human prekallikrein), but binds to the active formof plasma kallikrein (e.g., human plasma kallikrein).

In certain embodiments, the protein binds at or near the active site ofthe catalytic domain of plasma kallikrein, or a fragment thereof, orbinds an epitope that overlaps with the active site of plasmakallikrein.

In some embodiments, the protein binds to one or more amino acids thatform the catalytic triad of plasma kallikrein: His434, Asp483, and/orSer578 (numbering based on the human sequence). In other embodiments,the protein binds to one or more amino acids that form a region forsubstrate recognition: Arg551, Gln553, Tyr555, Thr558, and/or Arg560(numbering based on the human sequence). In some embodiments, the plasmakallikrein binding protein binds one or more amino acids of: S478, N481,S525, and K526 (numbering based on the human kallikrein sequence).

In some embodiments, the protein binds to one or more amino acids ofSer479, Tyr563, and/or Asp585 (numbering based on the human sequence).

In some embodiments, the plasma kallikrein binding protein decreasesFactor XIIa and/or bradykinin production by greater than about 5%, about10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%,about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about75%, about 80%, about 85%, about 90%, or about 95% as compared to astandard, e.g., the Factor XIIa and/or bradykinin production under thesame conditions but in the absence of the protein.

In some embodiments, the plasma kallikrein binding protein has anapparent inhibition constant (K_(i,app)) of less than 1000, 500, 100,10, 5, 1, 0.5 or 0.2 nM.

In one embodiment, the HC and LC variable domain sequences arecomponents of the same polypeptide chain.

In some embodiments, the plasma kallikrein binding protein has a serumresidence time of 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks or more, invivo, e.g., in humans. In one embodiment, the plasma kallikrein bindingprotein is an IgG, e.g., an IgG1, IgG2, IgG3 or IgG4, that has a serumresidence time of 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks or more invivo, e.g., in humans.

In some embodiments, the plasma kallikrein binding protein is physicallyassociated with a moiety that improves serum residence time, e.g., amoiety described herein.

In another embodiment, the HC and LC variable domain sequences arecomponents of different polypeptide chains. For example, the plasmakallikrein binding protein is an IgG, e.g., IgG1, IgG2, IgG3, or IgG4.The plasma kallikrein binding protein can be a soluble Fab (sFab).

In other implementations the plasma kallikrein binding protein includesa Fab2′, scFv, minibody, scFv::Fc fusion, Fab::HSA fusion, HSA::Fabfusion, Fab::HSA::Fab fusion, or other molecule that comprises theantigen combining site of one of the binding proteins herein. The VH andVL regions of these Fabs can be provided as IgG, Fab, Fab2, Fab2′, scFv,PEGylated Fab, PEGylated scFv, PEGylated Fab2, VH::CH1::HSA+LC,HSA::VH::CH1+LC, LC::HSA+VH::CH1, HSA::LC+VH::CH1, or other appropriateconstruction.

In one embodiment, the plasma kallikrein binding protein is a human orhumanized antibody or is non-immunogenic in a human. For example, theprotein includes one or more human antibody framework regions, e.g., allhuman framework regions.

In one embodiment, the plasma kallikrein binding protein includes ahuman Fc domain, or an Fc domain that is at least 95, 96, 97, 98, or 99%identical to a human Fc domain.

In one embodiment, the plasma kallikrein binding protein is a primate orprimatized antibody or is non-immunogenic in a human. For example, theprotein includes one or more primate antibody framework regions, e.g.,all primate framework regions.

In one embodiment, the plasma kallikrein binding protein includes aprimate Fc domain, or an Fc domain that is at least 95, 96, 97, 98, or99% identical to a primate Fc domain. “Primate” includes humans (Homosapiens), chimpanzees (Pan troglodytes and Pan paniscus (bonobos)),gorillas (Gorilla gorilla), gibons, monkeys, lemurs, aye-ayes(Daubentonia madagascariensis), and tarsiers.

In one embodiment, the plasma kallikrein binding protein includes humanframework regions, or framework regions that are at least 95, 96, 97,98, or 99% identical to human framework regions.

In certain embodiments, the plasma kallikrein binding protein includesno sequences from mice or rabbits (e.g., is not a murine or rabbitantibody).

In certain embodiments, the protein is capable of binding to a cell ortissue, e.g., that expresses plasma kallikrein.

In one embodiment, the plasma kallikrein binding protein is physicallyassociated with a nanoparticle, and can be used to guide a nanoparticleto a cell or tissue expressing plasma kallikrein.

In some aspects, the disclosure features a method promoting woundhealing in a subject, the method comprising:

administering an isolated protein (e.g., antibody, e.g., human antibody)comprising a heavy chain immunoglobulin variable domain sequence and alight chain immunoglobulin variable domain sequence to the subject,wherein:

the heavy chain immunoglobulin variable domain sequence comprises one,two, or three (e.g., three) CDR regions from the heavy chain variabledomain of a protein described herein, and/or

the light chain immunoglobulin variable domain sequence comprises one,two, or three (e.g., three) CDR regions from the light chain variabledomain of a protein described herein,

wherein the protein binds to plasma kallikrein.

In some embodiments, the protein is administered in combination withanother treatment for wound healing.

In some embodiments, the heavy chain immunoglobulin variable domainsequence comprises one, two, or three (e.g., three) CDR regions from theheavy chain variable domain of M162-A04, M160-G12, M142-H08, X63-G06,X101-A01, X81-B01, X67-D03, X67-G04, X115-B07, X115-D05, X115-E09,X115-H06, X115-A03, X115-D01, X115-F02, X115-G04, M29-D09, M145-D11,M06-D09 and M35-G04, and/or

the light chain immunoglobulin variable domain sequence comprises one,two, or three (e.g., three) CDR regions from the light chain variabledomain of M162-A04, M160-G12, M142-H08, X63-G06, X101-A01, X81-B01,X67-D03, X67-G04, X115-B07, X115-D05, X115-E09, X115-H06, X115-A03,X115-D01, X115-F02, X115-G04, M29-D09, M145-D11, M06-D09 and M35-G04(respectively).

In some embodiments, the protein inhibits plasma kallikrein.

In some embodiments, the one, two, or three (e.g., three) CDR regionsfrom the heavy chain variable domain are from X81-B01 and/or the one,two, or three (e.g., three) CDR regions from the light chain variabledomain are from X81-B01.

In some embodiments, the one, two, or three (e.g., three) CDR regionsfrom the heavy chain variable domain are from X67-D03 and/or the one,two, or three (e.g., three) CDR regions from the light chain variabledomain are from X67-D03.

In some embodiments, the heavy chain immunoglobulin variable domainsequence comprises the heavy chain variable domain of a proteindescribed herein, and/or the light chain immunoglobulin variable domainsequence comprises the light chain variable domain of a proteindescribed herein.

In some embodiments, the heavy chain immunoglobulin variable domainsequence comprises the heavy chain variable domain of M162-A04,M160-G12, M142-H08, X63-G06, X101-A01, X81-B01, X67-D03, X67-G04,X115-B07, X115-D05, X115-E09, X115-H06, X115-A03, X115-D01, X115-F02,X115-G04, M29-D09, M145-D11, M06-D09 and M35-G04, and/or the light chainimmunoglobulin variable domain sequence comprises the light chainvariable domain of M162-A04, M160-G12, M142-H08, X63-G06, X101-A01,X81-B01, X67-D03, X67-G04, X115-B07, X115-D05, X115-E09, X115-H06,X115-A03, X115-D01, X115-F02, X115-G04, M29-D09, M145-D11, M06-D09 andM35-G04 (respectively).

In some embodiments, the heavy chain immunoglobulin variable domainsequence comprises the heavy chain variable domain of X81-B01, and/orthe light chain immunoglobulin variable domain sequence comprises thelight chain variable domain of X81-B01.

In some embodiments, the heavy chain immunoglobulin variable domainsequence comprises the heavy chain variable domain of X67-D03, and/orthe light chain immunoglobulin variable domain sequence comprises thelight chain variable domain of X67-D03.

In some embodiments, the protein comprises the heavy chain of a proteindescribed herein, and/or the light chain of a protein described herein.

In some embodiments, the protein comprises the heavy chain of M162-A04,M160-G12, M142-H08, X63-G06, X101-A01, X81-B01, X67-D03, X67-G04,X115-B07, X115-D05, X115-E09, X115-H06, X115-A03, X115-D01, X115-F02,X115-G04, M29-D09, M145-D11, M06-D09 and M35-G04, and/or the light chainof M162-A04, M160-G12, M142-H08, X63-G06, X101-A01, X81-B01, X67-D03,X67-G04, X115-B07, X115-D05, X115-E09, X115-H06, X115-A03, X115-D01,X115-F02, X115-G04, M29-D09, M145-D11, M06-D09 and M35-G04(respectively).

In some embodiments, the protein comprises the heavy chain of X81-B01,and/or the light chain of X81-B01.

In some embodiments, the protein comprises the heavy chain of X67-D03,and/or the light chain of X67-D03.

In some embodiments, the plasma kallikrein binding protein does not bindprekallikrein (e.g., human prekallikrein and/or murine prekallikrein),but binds to the active form of plasma kallikrein (e.g., human plasmakallikrein and/or murine plasma kallikrein).

In some embodiments, the plasma kallikrein binding protein decreasesFactor XIIa and/or bradykinin production by greater than about 5%, about10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%,about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about75%, about 80%, about 85%, about 90%, or about 95% as compared to astandard, e.g., the Factor XIIa and/or bradykinin production under thesame conditions but in the absence of the protein.

In some embodiments, the protein includes one or more of the followingcharacteristics: (a) a human CDR or human framework region; (b) the HCimmunoglobulin variable domain sequence comprises one or more (e.g., 1,2, or 3) CDRs that are at least 85, 88, 89, 90, 91, 92, 93, 94, 95, 96,97, 98, 99, or 100% identical to a CDR of a HC variable domain describedherein; (c) the LC immunoglobulin variable domain sequence comprises oneor more (e.g., 1, 2, or 3) CDRs that are at least 85, 88, 89, 90, 91,92, 93, 94, 95, 96, 97, 98, 99, or 100% identical to a CDR of a LCvariable domain described herein; (d) the LC immunoglobulin variabledomain sequence is at least 85, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97,98, 99, or 100% identical to a LC variable domain described herein(e.g., overall or in framework regions or CDRs); (e) the HCimmunoglobulin variable domain sequence is at least 85, 88, 89, 90, 91,92, 93, 94, 95, 96, 97, 98, 99, or 100% identical to a HC variabledomain described herein (e.g., overall or in framework regions or CDRs);(f) the protein binds an epitope bound by a protein described herein, orcompetes for binding with a protein described herein; and (g) a primateCDR or primate framework region.

In some embodiments, the protein has an apparent inhibition constant(K_(i,app)) of less than 1000, 500, 100, 5, 1, 0.5 or 0.2 nM.

In some embodiments, the antibody does not bind prekallikrein (e.g.,human prekallikrein and/or murine prekallikrein), but binds to theactive form of plasma kallikrein (e.g., human plasma kallikrein and/ormurine plasma kallikrein).

In a preferred embodiment, the protein is an antibody (e.g., a humanantibody) having the light and heavy chains of antibodies selected fromthe group consisting of M162-A04, M160-G12, M142-H08, X63-G06, X101-A01,X81-B01, X67-D03, X67-G04, X115-B07, X115-D05, X115-E09, X115-H06,X115-A03, X115-D01, X115-F02, X115-G04, M29-D09, M145-D11, M06-D09 andM35-G04.

In a preferred embodiment, the protein is an antibody (e.g., a humanantibody) having the heavy chain of an antibody selected from the groupconsisting of: M162-A04, M160-G12, M142-H08, X63-G06, X101-A01, X81-B01,X67-D03, X67-G04, X115-B07, X115-D05, X115-E09, X115-H06, X115-A03,X115-D01, X115-F02, X115-G04, M29-D09, M145-D11, M06-D09 and M35-G04.

In a preferred embodiment, the protein is an antibody (e.g., a humanantibody) having the light chain of an antibody selected from the groupconsisting of: M162-A04, M160-G12, M142-H08, X63-G06, X101-A01, X81-B01,X67-D03, X67-G04, X115-B07, X115-D05, X115-E09, X115-H06, X115-A03,X115-D01, X115-F02, X115-G04, M29-D09, M145-D11, M06-D09 and M35-G04.

In a preferred embodiment, the protein is an antibody (e.g., a humanantibody) having light and heavy antibody variable regions of anantibody selected from the group consisting of M162-A04, M160-G12,M142-H08, X63-G06-X101-A01, X81-B01, X67-D03, X67-G04, X115-B07,X115-D05, X115-E09, X115-H06, X115-A03, X115-D01, X115-F02, X115-G04,M29-D09, M145-D11, M06-D09 and M35-G04.

In a preferred embodiment, the protein is an antibody (e.g., a humanantibody) having a heavy chain antibody variable region of an antibodyselected from the group consisting of: M162-A04, M160-G12, M142-H08,X63-G06, X101-A01, X81-B01, X67-D03, X67-G04, X115-B07, X115-D05,X115-E09, X115-H06, X115-A03, X115-D01, X115-F02, X115-G04, M29-D09,M145-D11, M06-D09 and M35-G04.

In a preferred embodiment, the protein is an antibody (e.g., a humanantibody) having a light chain antibody variable region of an antibodyselected from the group consisting of: M162-A04, M160-G12, M142-H08,X63-G06, X101-A01, X81-B01, X67-D03, X67-G04, X115-B07, X115-D05,X115-E09, X115-H06, X115-A03, X115-D01, X115-F02, X115-G04, M29-D09,M145-D11, M06-D09 and M35-G04.

In a preferred embodiment, the protein is an antibody (e.g., a humanantibody) having one or more (e.g., 1, 2, or 3) heavy chain CDRsselected from the corresponding CDRs of the group of heavy chainsconsisting of M162-A04, M160-G12, M142-H08, X63-G06, X101-A01, X81-B01,X67-D03, X67-G04, X115-B07, X115-D05, X115-E09, X115-H06, X115-A03,X115-D01, X115-F02, X115-G04, M29-D09, M145-D11, M06-D09 and M35-G04.

In a preferred embodiment, the protein is an antibody (e.g., a humanantibody) having one or more (e.g., 1, 2, or 3) light chain CDRsselected from the corresponding CDRs of the group of light chainsconsisting of M162-A04, M160-G12, M142-H08, X63-G06, X101-A01, X81-B01,X67-D03, X67-G04, X115-B07, X115-D05, X115-E09, X115-H06, X115-A03,X115-D01, X115-F02, X115-G04, M29-D09, M145-D11, M06-D09 and M35-G04.

In a preferred embodiment, the protein is an antibody (e.g., a humanantibody) having one or more (e.g., 1, 2, or 3) heavy chain CDRsselected from the corresponding CDRs of the group of heavy chainsconsisting of M162-A04, M160-G12, M142-H08, X63-G06, X101-A01, X81-B01,X67-D03, X67-G04, X115-B07, X115-D05, X115-E09, X115-H06, X115-A03,X115-D01, X115-F02, X115-G04, M29-D09, M145-D11, M06-D09 and M35-G04 andone or more (e.g., 1, 2, or 3) light chain CDRs selected from thecorresponding CDRs of the group of light chains consisting of M162-A04,M160-G12, M142-H08, X63-G06, X101-A01, X81-B01, X67-D03, X67-G04,X115-B07, X115-D05, X115-E09, X115-H06, X115-A03, X115-D01, X115-F02,X115-G04, M29-D09, M145-D11, M06-D09 and M35-G04 (respectively).

In a preferred embodiment, the protein is an antibody (e.g., a humanantibody) having the light and heavy chains of X81-B01.

In a preferred embodiment, the protein is an antibody (e.g., a humanantibody) having the heavy chain of X81-B01.

In a preferred embodiment, the protein is an antibody (e.g., a humanantibody) having the light chain of X81-B01.

In a preferred embodiment, the protein is an antibody (e.g., a humanantibody) having light and heavy antibody variable regions of anantibody selected from X81-B01.

In a preferred embodiment, the protein is an antibody (e.g., a humanantibody) having a heavy chain antibody variable region of X81-B01.

In a preferred embodiment, the protein is an antibody (e.g., a humanantibody) having a light chain antibody variable region of X81-B01.

In a preferred embodiment, the protein is an antibody (e.g., a humanantibody) having one or more (e.g., 1, 2, or 3) heavy chain CDRs fromthe corresponding CDRs of the heavy chain of X81-B01.

In a preferred embodiment, the protein is an antibody (e.g., a humanantibody) having one or more (e.g., 1, 2, or 3) light chain CDRs fromthe corresponding CDRs of the light chain of X81-B01.

In a preferred embodiment, the protein is an antibody (e.g., a humanantibody) having one or more (e.g., 1, 2, or 3) heavy chain CDRs fromthe heavy chain of X81-B01 and one or more (e.g., 1, 2, or 3) lightchain CDRs from the corresponding CDRs of the light chain of X81-B01.

In a preferred embodiment, the protein is an antibody (e.g., a humanantibody) having the light and heavy chains of X67-D03.

In a preferred embodiment, the protein is an antibody (e.g., a humanantibody) having the heavy chain of X67-D03.

In a preferred embodiment, the protein is an antibody (e.g., a humanantibody) having the light chain of X67-D03.

In a preferred embodiment, the protein is an antibody (e.g., a humanantibody) having light and heavy antibody variable regions of anantibody selected from X67-D03.

In a preferred embodiment, the protein is an antibody (e.g., a humanantibody) having a heavy chain antibody variable region of X67-D03.

In a preferred embodiment, the protein is an antibody (e.g., a humanantibody) having a light chain antibody variable region of X67-D03.

In a preferred embodiment, the protein is an antibody (e.g., a humanantibody) having one or more (e.g., 1, 2, or 3) heavy chain CDRs fromthe corresponding CDRs of the heavy chain of X67-D03.

In a preferred embodiment, the protein is an antibody (e.g., a humanantibody) having one or more (e.g., 1, 2, or 3) light chain CDRs fromthe corresponding CDRs of the light chain of X67-D03.

In a preferred embodiment, the protein is an antibody (e.g., a humanantibody) having one or more (e.g., 1, 2, or 3) heavy chain CDRs fromthe heavy chain of X67-D03 and one or more (e.g., 1, 2, or 3) lightchain CDRs from the corresponding CDRs of the light chain of X67-D03.

In one embodiment, the HC and LC variable domain sequences arecomponents of the same polypeptide chain.

In some embodiments, the plasma kallikrein binding protein has a serumresidence time of 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks or more, invivo, e.g., in humans. In one embodiment, the plasma kallikrein bindingprotein is an IgG, e.g., an IgG1, IgG2, IgG3 or IgG4, that has a serumresidence time of 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks or more invivo, e.g., in humans.

In some embodiments, the plasma kallikrein binding protein is physicallyassociated with a moiety that improves serum residence time, e.g., amoiety described herein.

In another embodiment, the HC and LC variable domain sequences arecomponents of different polypeptide chains. For example, the protein isan IgG, e.g., IgG1, IgG2, IgG3, or IgG4. The protein can be a solubleFab (sFab).

In other implementations the protein includes a Fab2′, scFv, minibody,scFv::Fc fusion, Fab::HSA fusion, HSA::Fab fusion, Fab::HSA::Fab fusion,or other molecule that comprises the antigen combining site of one ofthe binding proteins herein. The VH and VL regions of these Fabs can beprovided as IgG, Fab, Fab2, Fab2′, scFv, PEGylated Fab, PEGylated scFv,PEGylated Fab2, VH::CH1::HSA+LC, HSA::VH::CH1+LC, LC::HSA+VH::CH1,HSA::LC+VH::CH1, or other appropriate construction.

In one embodiment, the protein is a human or humanized antibody or isnon-immunogenic in a human. For example, the protein includes one ormore human antibody framework regions, e.g., all human frameworkregions.

In one embodiment, the protein includes a human Fc domain, or an Fcdomain that is at least 95, 96, 97, 98, or 99% identical to a human Fcdomain.

In one embodiment, the protein is a primate or primatized antibody or isnon-immunogenic in a human. For example, the protein includes one ormore primate antibody framework regions, e.g., all primate frameworkregions.

In one embodiment, the protein includes a primate Fc domain, or an Fcdomain that is at least 95, 96, 97, 98, or 99% identical to a primate Fcdomain. “Primate” includes humans (Homo sapiens), chimpanzees (Pantroglodytes and Pan paniscus (bonobos)), gorillas (Gorilla gorilla),gibons, monkeys, lemurs, aye-ayes (Daubentonia madagascariensis), andtarsiers.

In one embodiment, the protein includes human framework regions, orframework regions that are at least 95, 96, 97, 98, or 99% identical tohuman framework regions.

In certain embodiments, the protein includes no sequences from mice orrabbits (e.g., is not a murine or rabbit antibody).

In certain embodiments, the protein is capable of binding to a cell ortissue, e.g., that expresses plasma kallikrein.

In one embodiment, protein is physically associated with a nanoparticle,and can be used to guide a nanoparticle to a cell or tissue expressingplasma kallikrein.

In some aspects, the disclosure features a method of treating orpreventing rheumatoid arthritis in a subject, the method comprising:

administering an isolated protein (e.g., antibody, e.g., human antibody)comprising a heavy chain immunoglobulin variable domain sequence and alight chain immunoglobulin variable domain sequence to the subject,wherein:

the heavy chain immunoglobulin variable domain sequence comprises one,two, or three (e.g., three) CDR regions from the heavy chain variabledomain of a protein described herein, and/or

the light chain immunoglobulin variable domain sequence comprises one,two, or three (e.g., three) CDR regions from the light chain variabledomain of a protein described herein,

wherein the protein binds to plasma kallikrein.

In some embodiments, the plasma kallikrein binding protein does not bindprekallikrein (e.g., human prekallikrein and/or murine prekallikrein),but binds to the active form of plasma kallikrein (e.g., human plasmakallikrein and/or murine plasma kallikrein).

In some embodiments, the protein is administered in combination withanother treatment for rheumatoid arthritis.

In some embodiments, the protein inhibits plasma kallikrein (e.g., humanplasma kallikrein and/or murine plasma kallikrein).

In some embodiments, the heavy chain immunoglobulin variable domainsequence comprises the heavy chain variable domain of a proteindescribed herein, and/or the light chain immunoglobulin variable domainsequence comprises the light chain variable domain of a proteindescribed herein.

In some embodiments, the protein comprises the heavy chain of a proteindescribed herein, and/or the light chain of a protein described herein.

In some aspects, the disclosure features a method of treating orpreventing gout in a subject, the method comprising:

administering an isolated protein (e.g., antibody, e.g., human antibody)comprising a heavy chain immunoglobulin variable domain sequence and alight chain immunoglobulin variable domain sequence to the subject,wherein:

the heavy chain immunoglobulin variable domain sequence comprises one,two, or three (e.g., three) CDR regions from the heavy chain variabledomain of a protein described herein, and/or

the light chain immunoglobulin variable domain sequence comprises one,two, or three (e.g., three) CDR regions from the light chain variabledomain of a protein described herein,

wherein the protein binds to plasma kallikrein.

In some embodiments, the plasma kallikrein binding protein does not bindprekallikrein (e.g., human prekallikrein and/or murine prekallikrein),but binds to the active form of plasma kallikrein (e.g., human plasmakallikrein and/or murine plasma kallikrein).

In some embodiments, the protein is administered in combination withanother treatment for gout.

In some embodiments, the protein inhibits plasma kallikrein (e.g., humanplasma kallikrein and/or murine plasma kallikrein).

In some embodiments, the heavy chain immunoglobulin variable domainsequence comprises the heavy chain variable domain of a proteindescribed herein, and/or the light chain immunoglobulin variable domainsequence comprises the light chain variable domain of a proteindescribed herein.

In some embodiments, the protein comprises the heavy chain of a proteindescribed herein, and/or the light chain of a protein described herein.

In some aspects, the disclosure features a method of treating orpreventing intestinal bowel disease in a subject, the method comprising:

administering an isolated protein (e.g., antibody, e.g., human antibody)comprising a heavy chain immunoglobulin variable domain sequence and alight chain immunoglobulin variable domain sequence to the subject,wherein:

the heavy chain immunoglobulin variable domain sequence comprises one,two, or three (e.g., three) CDR regions from the heavy chain variabledomain of a protein described herein, and/or

the light chain immunoglobulin variable domain sequence comprises one,two, or three (e.g., three) CDR regions from the light chain variabledomain of a protein described herein,

wherein the protein binds to plasma kallikrein.

In some embodiments, the plasma kallikrein binding protein does not bindprekallikrein (e.g., human prekallikrein and/or murine prekallikrein),but binds to the active form of plasma kallikrein (e.g., human plasmakallikrein and/or murine plasma kallikrein).

In some embodiments, the protein is administered in combination withanother treatment for intestinal bowel disease.

In some embodiments, the protein inhibits plasma kallikrein (e.g., humanplasma kallikrein and/or murine plasma kallikrein).

In some embodiments, the heavy chain immunoglobulin variable domainsequence comprises the heavy chain variable domain of a proteindescribed herein, and/or the light chain immunoglobulin variable domainsequence comprises the light chain variable domain of a proteindescribed herein.

In some embodiments, the protein comprises the heavy chain of a proteindescribed herein, and/or the light chain of a protein described herein.

In some aspects, the disclosure features a method of treating orpreventing oral mucositis in a subject, the method comprising:

administering an isolated protein (e.g., antibody, e.g., human antibody)comprising a heavy chain immunoglobulin variable domain sequence and alight chain immunoglobulin variable domain sequence to the subject,wherein:

the heavy chain immunoglobulin variable domain sequence comprises one,two, or three (e.g., three) CDR regions from the heavy chain variabledomain of a protein described herein, and/or

the light chain immunoglobulin variable domain sequence comprises one,two, or three (e.g., three) CDR regions from the light chain variabledomain of a protein described herein,

wherein the protein binds to plasma kallikrein.

In some embodiments, the plasma kallikrein binding protein does not bindprekallikrein (e.g., human prekallikrein and/or murine prekallikrein),but binds to the active form of plasma kallikrein (e.g., human plasmakallikrein and/or murine plasma kallikrein).

In some embodiments, the protein is administered in combination withanother treatment for oral mucositis.

In some embodiments, the protein inhibits plasma kallikrein (e.g., humanplasma kallikrein and/or murine plasma kallikrein).

In some embodiments, the heavy chain immunoglobulin variable domainsequence comprises the heavy chain variable domain of a proteindescribed herein, and/or the light chain immunoglobulin variable domainsequence comprises the light chain variable domain of a proteindescribed herein.

In some embodiments, the protein comprises the heavy chain of a proteindescribed herein, and/or the light chain of a protein described herein.

In some aspects, the disclosure features a method of treating orpreventing neuropathic pain in a subject, the method comprising:

administering an isolated protein (e.g., antibody, e.g., human antibody)comprising a heavy chain immunoglobulin variable domain sequence and alight chain immunoglobulin variable domain sequence to the subject,wherein:

the heavy chain immunoglobulin variable domain sequence comprises one,two, or three (e.g., three) CDR regions from the heavy chain variabledomain of a protein described herein, and/or

the light chain immunoglobulin variable domain sequence comprises one,two, or three (e.g., three) CDR regions from the light chain variabledomain of a protein described herein,

wherein the protein binds to plasma kallikrein.

In some embodiments, the plasma kallikrein binding protein does not bindprekallikrein (e.g., human prekallikrein and/or murine prekallikrein),but binds to the active form of plasma kallikrein (e.g., human plasmakallikrein and/or murine plasma kallikrein).

In some embodiments, the protein is administered in combination withanother treatment for neuropathic pain.

In some embodiments, the protein inhibits plasma kallikrein (e.g., humanplasma kallikrein and/or murine plasma kallikrein).

In some embodiments, the heavy chain immunoglobulin variable domainsequence comprises the heavy chain variable domain of a proteindescribed herein, and/or the light chain immunoglobulin variable domainsequence comprises the light chain variable domain of a proteindescribed herein.

In some embodiments, the protein comprises the heavy chain of a proteindescribed herein, and/or the light chain of a protein described herein.

In some aspects, the disclosure features a method of treating orpreventing inflammatory pain in a subject, the method comprising:

administering an isolated protein (e.g., antibody, e.g., human antibody)comprising a heavy chain immunoglobulin variable domain sequence and alight chain immunoglobulin variable domain sequence to the subject,wherein:

the heavy chain immunoglobulin variable domain sequence comprises one,two, or three (e.g., three) CDR regions from the heavy chain variabledomain of a protein described herein, and/or

the light chain immunoglobulin variable domain sequence comprises one,two, or three (e.g., three) CDR regions from the light chain variabledomain of a protein described herein,

wherein the protein binds to plasma kallikrein.

In some embodiments, the plasma kallikrein binding protein does not bindprekallikrein (e.g., human prekallikrein and/or murine prekallikrein),but binds to the active form of plasma kallikrein (e.g., human plasmakallikrein and/or murine plasma kallikrein).

In some embodiments, the protein is administered in combination withanother treatment for inflammatory pain.

In some embodiments, the protein inhibits plasma kallikrein (e.g., humanplasma kallikrein and/or murine plasma kallikrein).

In some embodiments, the heavy chain immunoglobulin variable domainsequence comprises the heavy chain variable domain of a proteindescribed herein, and/or the light chain immunoglobulin variable domainsequence comprises the light chain variable domain of a proteindescribed herein.

In some embodiments, the protein comprises the heavy chain of a proteindescribed herein, and/or the light chain of a protein described herein.

In some aspects, the disclosure features a method of treating orpreventing spinal stenosis-degenerative spine disease in a subject, themethod comprising:

administering an isolated protein (e.g., antibody, e.g., human antibody)comprising a heavy chain immunoglobulin variable domain sequence and alight chain immunoglobulin variable domain sequence to the subject,wherein:

the heavy chain immunoglobulin variable domain sequence comprises one,two, or three (e.g., three) CDR regions from the heavy chain variabledomain of a protein described herein, and/or

the light chain immunoglobulin variable domain sequence comprises one,two, or three (e.g., three) CDR regions from the light chain variabledomain of a protein described herein,

wherein the protein binds to plasma kallikrein.

In some embodiments, the plasma kallikrein binding protein does not bindprekallikrein (e.g., human prekallikrein and/or murine prekallikrein),but binds to the active form of plasma kallikrein (e.g., human plasmakallikrein and/or murine plasma kallikrein).

In some embodiments, the protein is administered in combination withanother treatment for spinal stenosis-degenerative spine disease.

In some embodiments, the protein inhibits plasma kallikrein (e.g., humanplasma kallikrein and/or murine plasma kallikrein).

In some embodiments, the heavy chain immunoglobulin variable domainsequence comprises the heavy chain variable domain of a proteindescribed herein, and/or the light chain immunoglobulin variable domainsequence comprises the light chain variable domain of a proteindescribed herein.

In some embodiments, the protein comprises the heavy chain of a proteindescribed herein, and/or the light chain of a protein described herein.

In some aspects, the disclosure features a method of treating orpreventing arterial or venous thrombosis in a subject, the methodcomprising:

administering an isolated protein (e.g., antibody, e.g., human antibody)comprising a heavy chain immunoglobulin variable domain sequence and alight chain immunoglobulin variable domain sequence to the subject,wherein:

the heavy chain immunoglobulin variable domain sequence comprises one,two, or three (e.g., three) CDR regions from the heavy chain variabledomain of a protein described herein, and/or

the light chain immunoglobulin variable domain sequence comprises one,two, or three (e.g., three) CDR regions from the light chain variabledomain of a protein described herein,

wherein the protein binds to plasma kallikrein.

In some embodiments, the plasma kallikrein binding protein does not bindprekallikrein (e.g., human prekallikrein and/or murine prekallikrein),but binds to the active form of plasma kallikrein (e.g., human plasmakallikrein and/or murine plasma kallikrein).

In some embodiments, the protein is administered in combination withanother treatment for arterial or venous thrombosis.

In some embodiments, the protein inhibits plasma kallikrein (e.g., humanplasma kallikrein and/or murine plasma kallikrein).

In some embodiments, the heavy chain immunoglobulin variable domainsequence comprises the heavy chain variable domain of a proteindescribed herein, and/or the light chain immunoglobulin variable domainsequence comprises the light chain variable domain of a proteindescribed herein.

In some embodiments, the protein comprises the heavy chain of a proteindescribed herein, and/or the light chain of a protein described herein.

In some aspects, the disclosure features a method of treating orpreventing post operative ileus in a subject, the method comprising:

administering an isolated protein (e.g., antibody, e.g., human antibody)comprising a heavy chain immunoglobulin variable domain sequence and alight chain immunoglobulin variable domain sequence to the subject,wherein:

-   -   the heavy chain immunoglobulin variable domain sequence        comprises one, two, or three (e.g., three) CDR regions from the        heavy chain variable domain of a protein described herein,        and/or

the light chain immunoglobulin variable domain sequence comprises one,two, or three (e.g., three) CDR regions from the light chain variabledomain of a protein described herein,

wherein the protein binds to plasma kallikrein.

In some embodiments, the plasma kallikrein binding protein does not bindprekallikrein (e.g., human prekallikrein and/or murine prekallikrein),but binds to the active form of plasma kallikrein (e.g., human plasmakallikrein and/or murine plasma kallikrein).

In some embodiments, the protein is administered in combination withanother treatment for post operative ileus.

In some embodiments, the protein inhibits plasma kallikrein (e.g., humanplasma kallikrein and/or murine plasma kallikrein).

In some embodiments, the heavy chain immunoglobulin variable domainsequence comprises the heavy chain variable domain of a proteindescribed herein, and/or the light chain immunoglobulin variable domainsequence comprises the light chain variable domain of a proteindescribed herein.

In some embodiments, the protein comprises the heavy chain of a proteindescribed herein, and/or the light chain of a protein described herein.

In some aspects, the disclosure features a method of treating orpreventing aortic aneurysm in a subject, the method comprising:

administering an isolated protein (e.g., antibody, e.g., human antibody)comprising a heavy chain immunoglobulin variable domain sequence and alight chain immunoglobulin variable domain sequence to the subject,wherein:

the heavy chain immunoglobulin variable domain sequence comprises one,two, or three (e.g., three) CDR regions from the heavy chain variabledomain of a protein described herein, and/or

the light chain immunoglobulin variable domain sequence comprises one,two, or three (e.g., three) CDR regions from the light chain variabledomain of a protein described herein,

wherein the protein binds to plasma kallikrein.

In some embodiments, the plasma kallikrein binding protein does not bindprekallikrein (e.g., human prekallikrein and/or murine prekallikrein),but binds to the active form of plasma kallikrein (e.g., human plasmakallikrein and/or murine plasma kallikrein).

In some embodiments, the protein is administered in combination withanother treatment for aortic aneurysm.

In some embodiments, the protein inhibits plasma kallikrein (e.g., humanplasma kallikrein and/or murine plasma kallikrein).

In some embodiments, the heavy chain immunoglobulin variable domainsequence comprises the heavy chain variable domain of a proteindescribed herein, and/or the light chain immunoglobulin variable domainsequence comprises the light chain variable domain of a proteindescribed herein.

In some embodiments, the protein comprises the heavy chain of a proteindescribed herein, and/or the light chain of a protein described herein.

In some aspects, the disclosure features a method of treating orpreventing osteoarthritis in a subject, the method comprising:

administering an isolated protein (e.g., antibody, e.g., human antibody)comprising a heavy chain immunoglobulin variable domain sequence and alight chain immunoglobulin variable domain sequence to the subject,wherein:

the heavy chain immunoglobulin variable domain sequence comprises one,two, or three (e.g., three) CDR regions from the heavy chain variabledomain of a protein described herein, and/or

the light chain immunoglobulin variable domain sequence comprises one,two, or three (e.g., three) CDR regions from the light chain variabledomain of a protein described herein,

wherein the protein binds to plasma kallikrein.

In some embodiments, the plasma kallikrein binding protein does not bindprekallikrein (e.g., human prekallikrein and/or murine prekallikrein),but binds to the active form of plasma kallikrein (e.g., human plasmakallikrein and/or murine plasma kallikrein).

In some embodiments, the protein is administered in combination withanother treatment for osteoarthritis.

In some embodiments, the protein inhibits plasma kallikrein (e.g., humanplasma kallikrein and/or murine plasma kallikrein).

In some embodiments, the heavy chain immunoglobulin variable domainsequence comprises the heavy chain variable domain of a proteindescribed herein, and/or the light chain immunoglobulin variable domainsequence comprises the light chain variable domain of a proteindescribed herein.

In some embodiments, the protein comprises the heavy chain of a proteindescribed herein, and/or the light chain of a protein described herein.

In some aspects, the disclosure features a method of treating orpreventing vasculitis in a subject, the method comprising:

administering an isolated protein (e.g., antibody, e.g., human antibody)comprising a heavy chain immunoglobulin variable domain sequence and alight chain immunoglobulin variable domain sequence to the subject,wherein:

the heavy chain immunoglobulin variable domain sequence comprises one,two, or three (e.g., three) CDR regions from the heavy chain variabledomain of a protein described herein, and/or

the light chain immunoglobulin variable domain sequence comprises one,two, or three (e.g., three) CDR regions from the light chain variabledomain of a protein described herein,

wherein the protein binds to plasma kallikrein.

In some embodiments, the plasma kallikrein binding protein does not bindprekallikrein (e.g., human prekallikrein and/or murine prekallikrein),but binds to the active form of plasma kallikrein (e.g., human plasmakallikrein and/or murine plasma kallikrein).

In some embodiments, the protein is administered in combination withanother treatment for vasculitis.

In some embodiments, the protein inhibits plasma kallikrein (e.g., humanplasma kallikrein and/or murine plasma kallikrein).

In some embodiments, the heavy chain immunoglobulin variable domainsequence comprises the heavy chain variable domain of a proteindescribed herein, and/or the light chain immunoglobulin variable domainsequence comprises the light chain variable domain of a proteindescribed herein.

In some embodiments, the protein comprises the heavy chain of a proteindescribed herein, and/or the light chain of a protein described herein.

In some aspects, the disclosure features a method of treating orpreventing head trauma or peri-tumor brain edema in a subject, themethod comprising:

administering an isolated protein (e.g., antibody, e.g., human antibody)comprising a heavy chain immunoglobulin variable domain sequence and alight chain immunoglobulin variable domain sequence to the subject,wherein:

the heavy chain immunoglobulin variable domain sequence comprises one,two, or three (e.g., three) CDR regions from the heavy chain variabledomain of a protein described herein, and/or

the light chain immunoglobulin variable domain sequence comprises one,two, or three (e.g., three) CDR regions from the light chain variabledomain of a protein described herein,

wherein the protein binds to plasma kallikrein.

In some embodiments, the plasma kallikrein binding protein does not bindprekallikrein (e.g., human prekallikrein and/or murine prekallikrein),but binds to the active form of plasma kallikrein (e.g., human plasmakallikrein and/or murine plasma kallikrein).

In some embodiments, the protein is administered in combination withanother treatment for head trauma or peri-tumor brain edema.

In some embodiments, the protein inhibits plasma kallikrein (e.g., humanplasma kallikrein and/or murine plasma kallikrein).

In some embodiments, the heavy chain immunoglobulin variable domainsequence comprises the heavy chain variable domain of a proteindescribed herein, and/or the light chain immunoglobulin variable domainsequence comprises the light chain variable domain of a proteindescribed herein.

In some embodiments, the protein comprises the heavy chain of a proteindescribed herein, and/or the light chain of a protein described herein.

In some aspects, the disclosure features a method of treating orpreventing sepsis in a subject, the method comprising:

administering an isolated protein (e.g., antibody, e.g., human antibody)comprising a heavy chain immunoglobulin variable domain sequence and alight chain immunoglobulin variable domain sequence to the subject,wherein:

the heavy chain immunoglobulin variable domain sequence comprises one,two, or three (e.g., three) CDR regions from the heavy chain variabledomain of a protein described herein, and/or

the light chain immunoglobulin variable domain sequence comprises one,two, or three (e.g., three) CDR regions from the light chain variabledomain of a protein described herein,

wherein the protein binds to plasma kallikrein.

In some embodiments, the plasma kallikrein binding protein does not bindprekallikrein (e.g., human prekallikrein and/or murine prekallikrein),but binds to the active form of plasma kallikrein (e.g., human plasmakallikrein and/or murine plasma kallikrein).

In some embodiments, the protein is administered in combination withanother treatment for sepsis.

In some embodiments, the protein inhibits plasma kallikrein (e.g., humanplasma kallikrein and/or murine plasma kallikrein).

In some embodiments, the heavy chain immunoglobulin variable domainsequence comprises the heavy chain variable domain of a proteindescribed herein, and/or the light chain immunoglobulin variable domainsequence comprises the light chain variable domain of a proteindescribed herein.

In some embodiments, the protein comprises the heavy chain of a proteindescribed herein, and/or the light chain of a protein described herein.

In some aspects, the disclosure features a method of treating orpreventing acute middle cerebral artery (MCA) ischemic event (stroke) ina subject, the method comprising:

administering an isolated protein (e.g., antibody, e.g., human antibody)comprising a heavy chain immunoglobulin variable domain sequence and alight chain immunoglobulin variable domain sequence to the subject,wherein:

the heavy chain immunoglobulin variable domain sequence comprises one,two, or three (e.g., three) CDR regions from the heavy chain variabledomain of a protein described herein, and/or

the light chain immunoglobulin variable domain sequence comprises one,two, or three (e.g., three) CDR regions from the light chain variabledomain of a protein described herein, wherein the protein binds toplasma kallikrein.

In some embodiments, the plasma kallikrein binding protein does not bindprekallikrein (e.g., human prekallikrein and/or murine prekallikrein),but binds to the active form of plasma kallikrein (e.g., human plasmakallikrein and/or murine plasma kallikrein).

In some embodiments, the protein is administered in combination withanother treatment for acute middle cerebral artery (MCA) ischemic event(stroke).

In some embodiments, the protein inhibits plasma kallikrein (e.g., humanplasma kallikrein and/or murine plasma kallikrein).

In some embodiments, the heavy chain immunoglobulin variable domainsequence comprises the heavy chain variable domain of a proteindescribed herein, and/or the light chain immunoglobulin variable domainsequence comprises the light chain variable domain of a proteindescribed herein.

In some embodiments, the protein comprises the heavy chain of a proteindescribed herein, and/or the light chain of a protein described herein.

In some aspects, the disclosure features a method of treating orpreventing restenosis (e.g., after angioplasty) in a subject, the methodcomprising:

administering an isolated protein (e.g., antibody, e.g., human antibody)comprising a heavy chain immunoglobulin variable domain sequence and alight chain immunoglobulin variable domain sequence to the subject,wherein:

the heavy chain immunoglobulin variable domain sequence comprises one,two, or three (e.g., three) CDR regions from the heavy chain variabledomain of a protein described herein, and/or

the light chain immunoglobulin variable domain sequence comprises one,two, or three (e.g., three) CDR regions from the light chain variabledomain of a protein described herein,

wherein the protein binds to plasma kallikrein.

In some embodiments, the plasma kallikrein binding protein does not bindprekallikrein (e.g., human prekallikrein and/or murine prekallikrein),but binds to the active form of plasma kallikrein (e.g., human plasmakallikrein and/or murine plasma kallikrein).

In some embodiments, the protein is administered in combination withanother treatment for restenosis (e.g., after angioplasty).

In some embodiments, the protein inhibits plasma kallikrein (e.g., humanplasma kallikrein and/or murine plasma kallikrein).

In some embodiments, the heavy chain immunoglobulin variable domainsequence comprises the heavy chain variable domain of a proteindescribed herein, and/or the light chain immunoglobulin variable domainsequence comprises the light chain variable domain of a proteindescribed herein.

In some embodiments, the protein comprises the heavy chain of a proteindescribed herein, and/or the light chain of a protein described herein.

In some aspects, the disclosure features a method of treating orpreventing systemic lupus erythematosis nephritis in a subject, themethod comprising:

administering an isolated protein (e.g., antibody, e.g., human antibody)comprising a heavy chain immunoglobulin variable domain sequence and alight chain immunoglobulin variable domain sequence to the subject,wherein:

the heavy chain immunoglobulin variable domain sequence comprises one,two, or three (e.g., three) CDR regions from the heavy chain variabledomain of a protein described herein, and/or

the light chain immunoglobulin variable domain sequence comprises one,two, or three (e.g., three) CDR regions from the light chain variabledomain of a protein described herein,

wherein the protein binds to plasma kallikrein.

In some embodiments, the plasma kallikrein binding protein does not bindprekallikrein (e.g., human prekallikrein and/or murine prekallikrein),but binds to the active form of plasma kallikrein (e.g., human plasmakallikrein and/or murine plasma kallikrein).

In some embodiments, the protein is administered in combination withanother treatment for systemic lupus erythematosis nephritis.

In some embodiments, the protein inhibits plasma kallikrein (e.g., humanplasma kallikrein and/or murine plasma kallikrein).

In some embodiments, the heavy chain immunoglobulin variable domainsequence comprises the heavy chain variable domain of a proteindescribed herein, and/or the light chain immunoglobulin variable domainsequence comprises the light chain variable domain of a proteindescribed herein.

In some embodiments, the protein comprises the heavy chain of a proteindescribed herein, and/or the light chain of a protein described herein.

In some aspects, the disclosure features a method of treating orpreventing burn injury in a subject, the method comprising:

administering an isolated protein (e.g., antibody, e.g., human antibody)comprising a heavy chain immunoglobulin variable domain sequence and alight chain immunoglobulin variable domain sequence to the subject,wherein:

the heavy chain immunoglobulin variable domain sequence comprises one,two, or three (e.g., three) CDR regions from the heavy chain variabledomain of a protein described herein, and/or

the light chain immunoglobulin variable domain sequence comprises one,two, or three (e.g., three) CDR regions from the light chain variabledomain of a protein described herein,

wherein the protein binds to plasma kallikrein.

In some embodiments, the plasma kallikrein binding protein does not bindprekallikrein (e.g., human prekallikrein and/or murine prekallikrein),but binds to the active form of plasma kallikrein (e.g., human plasmakallikrein and/or murine plasma kallikrein).

In some embodiments, the protein is administered in combination withanother treatment for burn injury.

In some embodiments, the protein inhibits plasma kallikrein (e.g., humanplasma kallikrein and/or murine plasma kallikrein).

In some embodiments, the heavy chain immunoglobulin variable domainsequence comprises the heavy chain variable domain of a proteindescribed herein, and/or the light chain immunoglobulin variable domainsequence comprises the light chain variable domain of a proteindescribed herein.

In some embodiments, the protein comprises the heavy chain of a proteindescribed herein, and/or the light chain of a protein described herein.

In some aspects, the disclosure features a method of detecting plasmakallikrein in a sample, the method comprising: contacting the samplewith a plasma kallikrein binding protein (e.g., a plasma kallikreinbinding protein described herein); and detecting an interaction betweenthe protein and the plasma kallikrein, if present.

In some embodiments, the protein includes a detectable label.

In some embodiments, the plasma kallikrein binding protein does not bindprekallikrein (e.g., human prekallikrein and/or murine prekallikrein),but binds to the active form of plasma kallikrein (e.g., human plasmakallikrein and/or murine plasma kallikrein). In some embodiments, theplasma kallikrein binding protein binds prekallikrein (e.g., humanprekallikrein and/or murine prekallikrein) and the active form of plasmakallikrein (e.g., human plasma kallikrein and/or murine plasmakallikrein).

In some aspects, the disclosure features a method of detecting plasmakallikrein in a subject, the method comprising: administering a plasmakallikrein binding protein (e.g., a plasma kallikrein binding proteindescribed herein) to a subject; and detecting an interaction between theprotein and the plasma kallikrein in the subject, if present. Forexample, the detecting comprises imaging the subject.

In some embodiments, the protein further includes a detectable label.

In some embodiments, the plasma kallikrein binding protein does not bindprekallikrein (e.g., human prekallikrein and/or murine prekallikrein),but binds to the active form of plasma kallikrein (e.g., human plasmakallikrein and/or murine plasma kallikrein). In some embodiments, theplasma kallikrein binding protein binds prekallikrein (e.g., humanprekallikrein and/or murine prekallikrein) and the active form of plasmakallikrein (e.g., human plasma kallikrein and/or murine plasmakallikrein).

In some aspects, the disclosure features a method of modulating plasmakallikrein activity, e.g., in a method of treating or preventing aplasma kallikrein associated disorder. The method includes: contactingplasma kallikrein with a plasma kallikrein binding protein (e.g., aplasma kallikrein binding protein described herein) (e.g., in a humansubject), thereby modulating plasma kallikrein activity.

In some embodiments, the plasma kallikrein binding protein does not bindprekallikrein (e.g., human prekallikrein and/or murine prekallikrein),but binds to the active form of plasma kallikrein (e.g., human plasmakallikrein and/or murine plasma kallikrein).

In some embodiments, the plasma kallikrein associated disorder isselected from the group consisting of rheumatoid arthritis, gout,intestinal bowel disease, oral mucositis, neuropathic pain, inflammatorypain, spinal stenosis-degenerative spine disease, arterial or venousthrombosis, post operative ileus, aortic aneurysm, osteoarthritis,vasculitis, edema, hereditary angioedema, cerebral edema, pulmonaryembolism, stroke, clotting induced by ventricular assistance devices orstents, head trauma or peri-tumor brain edema, sepsis, acute middlecerebral artery (MCA) ischemic event (stroke), restenosis (e.g., afterangioplasty), systemic lupus erythematosis nephritis/vasculitis, andburn injury.

In some embodiments, the plasma kallikrein binding protein reducesabberent clotting associated with the contact activation system (i.e.,intrinsic activation system) by at least 10% as measured by e.g., anAPTT clotting assay. In other embodiments, the plasma kallikrein bindingprotein reduces abberent clotting associated with the contact activationsystem by at least 20%, at least 30%, at least 40%, at least 50%, atleast 60%, at least 70%, at least 80%, at least 90%, at least 95%, atleast 99%, or even 100% (i.e., no detectable abberent clotting).

In some aspects, the disclosure features a method of treating a plasmakallikrein associated disorder, the method comprising administering, toa subject, a plasma kallikrein binding protein (e.g., a plasmakallikrein binding protein described herein) in an amount sufficient totreat a plasma kallikrein associated disorder in the subject. The methodcan further include providing to the subject a second therapy that istherapy for the plasma kallikrein associated disorder, e.g., asdescribed herein.

In some embodiments, the plasma kallikrein associated disorder isselected from the group consisting of rheumatoid arthritis, gout,intestinal bowel disease, oral mucositis, neuropathic pain, inflammatorypain, spinal stenosis-degenerative spine disease, arterial or venousthrombosis, post operative ileus, aortic aneurysm, osteoarthritis,vasculitis, edema, hereditary angioedema, cerebral edema, pulmonaryembolism, stroke, clotting induced by ventricular assistance devices orstents, head trauma or peri-tumor brain edema, sepsis, acute middlecerebral artery (MCA) ischemic event (stroke), restenosis (e.g., afterangioplasty), systemic lupus erythematosis nephritis/vasculitis, andburn injury.

In some aspects, the disclosure features a method of imaging a subject.The method includes administering a plasma kallikrein binding protein(e.g., a plasma kallikrein binding protein described herein) to thesubject, and e.g., detecting an interaction between the protein and theplasma kallikrein in the subject, if present.

In some embodiments, the plasma kallikrein binding protein does not bindprekallikrein (e.g., human prekallikrein and/or murine prekallikrein),but binds to the active form of plasma kallikrein (e.g., human plasmakallikrein and/or murine plasma kallikrein). In some embodiments, theplasma kallikrein binding protein binds prekallikrein (e.g., humanprekallikrein and/or murine prekallikrein) and the active form of plasmakallikrein (e.g., human plasma kallikrein and/or murine plasmakallikrein).

In some embodiments, the protein does not inhibit plasma kallikreinactivity.

In some embodiments, the protein inhibits plasma kallikrein activity(e.g., human plasma kallikrein and/or murine plasma kallikrein).

In some embodiments, the plasma kallikrein binding protein may include adetectable label (e.g., a radionuclide or an MRI-detectable label).

In some embodiments, the subject has or is suspected of having a plasmakallikrein associated disorder. The method is useful, e.g., fordiagnosis of a plasma kallikrein associated disorder.

In some embodiments, the plasma kallikrein associated disorder isselected from the group consisting of rheumatoid arthritis, gout,intestinal bowel disease, oral mucositis, neuropathic pain, inflammatorypain, spinal stenosis-degenerative spine disease, arterial or venousthrombosis, post operative ileus, aortic aneurysm, osteoarthritis,vasculitis, edema, hereditary angioedema, cerebral edema, pulmonaryembolism, stroke, clotting induced by ventricular assistance devices orstents, head trauma or peri-tumor brain edema, sepsis, acute middlecerebral artery (MCA) ischemic event (stroke), restenosis (e.g., afterangioplasty), systemic lupus erythematosis nephritis, and burn injury.

In some embodiments, the plasma kallikrein binding protein reducesabberent clotting associated with the contact activation system (i.e.,intrinsic activation system) by at least 10% as measured by e.g., anAPTT clotting assay (e.g., by at least 20%, at least 30%, at least 40%,at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, atleast 95%, at least 99%, or even 100% (i.e., no detectable abberentclotting)).

In some aspects, the disclosure features a method of imaging plasmakallikrein, e.g., in a subject or sample (e.g., biopsy sample). Themethod includes administering a plasma kallikrein binding protein (e.g.,a plasma kallikrein binding protein described herein), e.g., to thesubject or the sample, and detecting an interaction between the proteinand the plasma kallikrein, if present.

In some embodiments, the plasma kallikrein binding protein does not bindprekallikrein (e.g., human prekallikrein and/or murine prekallikrein),but binds to the active form of plasma kallikrein (e.g., human plasmakallikrein and/or murine plasma kallikrein). In some embodiments, theplasma kallikrein binding protein binds prekallikrein (e.g., humanprekallikrein and/or murine prekallikrein) and the active form of plasmakallikrein (e.g., human plasma kallikrein and/or murine plasmakallikrein).

In some embodiments, the protein does not inhibit plasma kallikreinactivity.

In some embodiments, the protein inhibits plasma kallikrein activity(e.g., human plasma kallikrein and/or murine plasma kallikrein).

In some embodiments, the plasma kallikrein binding protein may include adetectable label (e.g., a radionuclide or an MRI-detectable label).

In some embodiments, the subject has or is suspected of having a plasmakallikrein associated disorder. The method is useful, e.g., fordiagnosis of a plasma kallikrein associated disorder.

In some embodiments, the plasma kallikrein associated disorder isselected from the group consisting of rheumatoid arthritis, gout,intestinal bowel disease, oral mucositis, neuropathic pain, inflammatorypain, spinal stenosis-degenerative spine disease, arterial or venousthrombosis, post operative ileus, aortic aneurysm, osteoarthritis,vasculitis, edema, hereditary angioedema, cerebral edema, pulmonaryembolism, stroke, clotting induced by ventricular assistance devices orstents, head trauma or peri-tumor brain edema, sepsis, acute middlecerebral artery (MCA) ischemic event (stroke), restenosis (e.g., afterangioplasty), systemic lupus erythematosis nephritis, and burn injury.

In some embodiments, the plasma kallikrein binding protein reducesabberent clotting associated with the contact activation system (i.e.,intrinsic activation system) by at least 10% as measured by e.g., anAPTT clotting assay (e.g., by at least 20%, at least 30%, at least 40%,at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, atleast 95%, at least 99%, or even 100% (i.e., no detectable abberentclotting)).

In one aspect, the disclosure features the use of a plasma kallikreinbinding protein described herein for the treatment of a disorderdescribed herein, e.g., rheumatoid arthritis, gout, intestinal boweldisease, oral mucositis, neuropathic pain, inflammatory pain, spinalstenosis-degenerative spine disease, arterial or venous thrombosis, postoperative ileus, aortic aneurysm, osteoarthritis, vasculitis, edema,hereditary angioedema, cerebral edema, pulmonary embolism, stroke,clotting induced by ventricular assistance devices or stents, headtrauma or peri-tumor brain edema, sepsis, acute middle cerebral artery(MCA) ischemic event (stroke), restenosis (e.g., after angioplasty),systemic lupus erythematosis nephritis, or burn injury; or to promotewound healing.

In some embodiments, the plasma kallikrein binding protein does not bindprekallikrein (e.g., human prekallikrein and/or murine prekallikrein),but binds to the active form of plasma kallikrein (e.g., human plasmakallikrein and/or murine plasma kallikrein). In some embodiments, theplasma kallikrein binding protein binds prekallikrein (e.g., humanprekallikrein and/or murine prekallikrein) and the active form of plasmakallikrein (e.g., human plasma kallikrein and/or murine plasmakallikrein).

In one aspect, the disclosure features the use of a plasma kallikreinbinding protein described herein for the manufacture of a medicament forthe treatment of a disorder described herein, e.g., rheumatoidarthritis, gout, intestinal bowel disease, oral mucositis, neuropathicpain, inflammatory pain, spinal stenosis-degenerative spine disease,arterial or venous thrombosis, post operative ileus, aortic aneurysm,osteoarthritis, vasculitis, edema, hereditary angioedema, cerebraledema, pulmonary embolism, stroke, clotting induced by ventricularassistance devices or stents, head trauma or peri-tumor brain edema,sepsis, acute middle cerebral artery (MCA) ischemic event (stroke),restenosis (e.g., after angioplasty), systemic lupus erythematosisnephritis, or burn injury; or for the manufacture of a medicament forwound healing.

In some embodiments, the plasma kallikrein binding protein reducesabberent clotting associated with the contact activation system (i.e.,intrinsic activation system) by at least 10% as measured by e.g., anAPTT clotting assay (e.g., by at least 20%, at least 30%, at least 40%,at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, atleast 95%, at least 99%, or even 100% (i.e., no detectable abberentclotting)).

In some embodiments, the plasma kallikrein binding protein does not bindprekallikrein (e.g., human prekallikrein and/or murine prekallikrein),but binds to the active form of plasma kallikrein (e.g., human plasmakallikrein and/or murine plasma kallikrein).

The details of one or more embodiments of the invention are set forth inthe accompanying drawings and the description below. Other features,objects, and advantages of the invention will be apparent from thedescription and drawings, and from the claims.

The contents of all cited references including literature references,issued patents, published or non-published patent applications citedthroughout this application as well as those listed below are herebyexpressly incorporated by reference in their entireties. In case ofconflict, the present application, including any definitions herein,will control.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a schematic representation of the role of plasma kallikrein(pKal) in intrinsic coagulation pathway and inflammation.

FIG. 2 depicts the effect of M162-A04 on carrageenan-induced rat pawedema. Paw swelling was measured by water displacement.

FIG. 3 depicts the effect of M162-A04 on carrageenan-induced thermalhyperalgesia. Pain latency was measured by the Hargreaves method aftercarrageenan injection.

FIG. 4 depicts the alignment of the light chain DNA sequence ofnongermlined (X63-G06) (SEQ ID NO:1991) and germlined, codon optimized(X81-B01) (SEQ ID NO:1992) versions of the same antibody discoveredusing ROLIC affinity maturation. Positions indicated with an asterisk(*) are conserved, whereas blank spaces correspond to bases changed inX81-B01 due to either codon optimization or germlining.

FIG. 5 depicts the alignment of the light chain amino acid sequence ofnongermlined (X63-G06) (SEQ ID NO:1993) and germlined, codon optimized(X81-B01) (SEQ ID NO:1994) versions of the same antibody discoveredusing ROLIC affinity maturation. Positions indicated with an asterisk(*) are conserved, whereas blank spaces correspond to amino acidschanged in X81-B01 due to germlining. A total of 11 amino acids differbetween the nongermlined (X63-G06) and germlined, codon optimizedantibody (X81-B01).

FIG. 6 depicts the alignment of the heavy chain DNA sequence ofnongermlined (X63-G06) (SEQ ID NO:1996) and germlined, codon optimized(X81-B01) (SEQ ID NO:1995) versions of the same antibody discoveredusing ROLIC affinity maturation. Positions indicated with an asterisk(*) are conserved, whereas blank spaces correspond to DNA bases changedin X81-B01 due to codon optimization.

FIG. 7 depicts the alignment of the heavy chain amino acid sequence ofnongermlined (X63-G06) (SEQ ID NO:1998) and germlined, codon optimized(X81-B01) (SEQ ID NO:1997) versions of the same antibody discoveredusing ROLIC affinity maturation. Positions indicated with an asterisk(*) are conserved. The two antibodies have the same amino acid sequencein the heavy chain.

FIG. 8A depicts the EPI-KAL2 competition for X81-B01 binding pKal.X81-B01 (IgG) was captured on an anti-human Fc fragment specific surfaceof a CM5 BIACORE® chip. pKal (100 nM) was flowed over the surface in thepresence (lower sensorgram in the figure) or absence of 1 μM EPI-KAL2(upper sensorgram in the figure).

FIG. 8B depicts the EPI-KAL2 competition for X67-D03 binding pKal.X67-D03 (IgG) was captured on an anti-human Fc fragment specific surfaceof a CM5 Biacore chip. pKal (100 nM) was flowed over the surface in thepresence (lower sensorgram in the figure) or absence of 1 μM EPI-KAL2(upper sensorgram in the figure).

FIG. 9 depicts the results of CLIPS epitope mapping for antibodieslisted in Table 12.

FIGS. 10A-10C depict ClustalW alignment of pKal sequences from differentspecies (SEQ ID NOs:1999-2006). Positions indicated by a “*” areconserved positions between, whereas positions indicated “:” indicateconservative substitutions between species. Positions indicated by a “.”have nonconservative substitutions in some species. Stretches of aminoacids indicated by the symbol “@” were shown to be highly solventexposed by solvent accessible surface area calculation. Stretches ofamino acids indicated by a “+” were identified as potential epitopes ofantibodies listed in Table 12. Amino acids highlighted in grey werefound by solvent accessible surface area calculation to be buried whencomplexed with a Kunitz domain active site inhibitor. The underlinedpositions are the amino acids that form the catalytic triad (His434,Asp483, and Ser578, numbering based on the human sequence).

FIGS. 11A and 11B depict a Biacore competition analysis with epi-kal2,as described herein in Example 12, for (i) DX-2922, and (ii) M6-D09antibodies.

FIG. 12 depicts a Biacore competition analysis with AEBSF, as describedherein in Example 12, for (i) DX-2911, and (ii) M6-D09 antibodies.

FIG. 13 depicts a Biocore analysis showing that DX-2922 binds to plasmakallikrein that bound to high molecular weight kininogen (HMWK).

FIG. 14 depicts a graph showing dose dependent inhibition of edema byX101-A01 in carrageenan-induced paw edema (CPE) in rats.

DETAILED DESCRIPTION Definitions

For convenience, before further description of the present invention,certain terms employed in the specification, examples and appendedclaims are defined here. Other terms are defined as they appear in thespecification.

The singular forms “a”, “an”, and “the” include plural references unlessthe context clearly dictates otherwise.

The term “agonist,” as used herein, is meant to refer to an agent thatmimics or up-regulates (e.g., potentiates or supplements) thebioactivity of a protein. An agonist can be a wild-type protein orderivative thereof having at least one bioactivity of the wild-typeprotein. An agonist can also be a compound which increases at least onebioactivity of a protein. An agonist can also be a compound whichincreases the interaction of a polypeptide with another molecule, e.g.,a target peptide or nucleic acid.

“Antagonist” as used herein is meant to refer to an agent thatdownregulates (e.g., suppresses or inhibits) at least one bioactivity ofa protein. An antagonist can be a compound which inhibits or decreasesthe interaction between a protein and another molecule, e.g., a targetpeptide or enzyme substrate. An antagonist can also be a compound whichreduces the amount of expressed protein present.

The term “antibody” refers to a protein that includes at least oneimmunoglobulin variable domain (variable region) or immunoglobulinvariable domain (variable region) sequence. For example, an antibody caninclude a heavy (H) chain variable region (abbreviated herein as VH orHV), and a light (L) chain variable region (abbreviated herein as VL orLV). In another example, an antibody includes two heavy (H) chainvariable regions and two light (L) chain variable regions. The term“antibody” encompasses antigen-binding fragments of antibodies (e.g.,single chain antibodies, Fab and sFab fragments, F(ab′)₂, Fd fragments,Fv fragments, scFv, and domain antibodies (dAb) fragments (de Wildt etal., Eur J Immunol. 1996; 26(3):629-39)) as well as complete antibodies.An antibody can have the structural features of IgA, IgG, IgE, IgD, IgM(as well as subtypes thereof). Antibodies may be from any source, butprimate (human and non-human primate) and primatized are preferred.

The VH and VL regions can be further subdivided into regions ofhypervariability, termed “complementarity determining regions” (“CDRs”),interspersed with regions that are more conserved, termed “frameworkregions” (“FRs”). The extent of the framework region and CDRs have beendefined (see, Kabat, E. A., et al. (1991) Sequences of Proteins ofImmunological Interest, Fifth Edition, U.S. Department of Health andHuman Services, NIH Publication No. 91-3242, and Chothia, C. et al.(1987) J. Mol. Biol. 196:901-917). Kabat definitions are used herein.Each VH and VL is typically composed of three CDRs and four FRs,arranged from amino-terminus to carboxy-terminus in the following order:FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

As used herein, an “immunoglobulin variable domain sequence” refers toan amino acid sequence which can form the structure of an immunoglobulinvariable domain such that one or more CDR regions are positioned in aconformation suitable for an antigen binding site. For example, thesequence may include all or part of the amino acid sequence of anaturally-occurring variable domain. For example, the sequence may omitone, two or more N- or C-terminal amino acids, internal amino acids, mayinclude one or more insertions or additional terminal amino acids, ormay include other alterations. In one embodiment, a polypeptide thatincludes immunoglobulin variable domain sequence can associate withanother immunoglobulin variable domain sequence to form an antigenbinding site, e.g., a structure that preferentially interacts withplasma kallikrein.

The VH or VL chain of the antibody can further include all or part of aheavy or light chain constant region, to thereby form a heavy or lightimmunoglobulin chain, respectively. In one embodiment, the antibody is atetramer of two heavy immunoglobulin chains and two light immunoglobulinchains, wherein the heavy and light immunoglobulin chains areinter-connected by, e.g., disulfide bonds. In IgGs, the heavy chainconstant region includes three immunoglobulin domains, CH1, CH2 and CH3.The light chain constant region includes a CL domain. The variableregion of the heavy and light chains contains a binding domain thatinteracts with an antigen. The constant regions of the antibodiestypically mediate the binding of the antibody to host tissues orfactors, including various cells of the immune system (e.g., effectorcells) and the first component (Clq) of the classical complement system.The light chains of the immunoglobulin may be of types kappa or lambda.In one embodiment, the antibody is glycosylated. An antibody can befunctional for antibody-dependent cytotoxicity and/orcomplement-mediated cytotoxicity.

One or more regions of an antibody can be human or effectively human.For example, one or more of the variable regions can be human oreffectively human. For example, one or more of the CDRs can be human,e.g., HC CDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2, and/or LC CDR3. Eachof the light chain (LC) and/or heavy chain (HC) CDRs can be human. HCCDR3 can be human. One or more of the framework regions can be human,e.g., FR1, FR2, FR3, and/or FR4 of the HC and/or LC. For example, the Fcregion can be human. In one embodiment, all the framework regions arehuman, e.g., derived from a human somatic cell, e.g., a hematopoieticcell that produces immunoglobulins or a non-hematopoietic cell. In oneembodiment, the human sequences are germline sequences, e.g., encoded bya germline nucleic acid. In one embodiment, the framework (FR) residuesof a selected Fab can be converted to the amino-acid type of thecorresponding residue in the most similar primate germline gene,especially the human germline gene. One or more of the constant regionscan be human or effectively human. For example, at least 70, 75, 80, 85,90, 92, 95, 98, or 100% of an immunoglobulin variable domain, theconstant region, the constant domains (CH1, CH2, CH3, and/or CL1), orthe entire antibody can be human or effectively human.

All or part of an antibody can be encoded by an immunoglobulin gene or asegment thereof. Exemplary human immunoglobulin genes include the kappa,lambda, alpha (IgA1 and IgA2), gamma (IgG1, IgG2, IgG3, IgG4), delta,epsilon and mu constant region genes, as well as the many immunoglobulinvariable region genes. Full-length immunoglobulin “light chains” (about25 KDa or about 214 amino acids) are encoded by a variable region geneat the NH2-terminus (about 110 amino acids) and a kappa or lambdaconstant region gene at the COOH-terminus. Full-length immunoglobulin“heavy chains” (about 50 KDa or about 446 amino acids), are similarlyencoded by a variable region gene (about 116 amino acids) and one of theother aforementioned constant region genes, e.g., gamma (encoding about330 amino acids). The length of human HC varies considerably because HCCDR3 varies from about 3 amino-acid residues to over 35 amino-acidresidues.

The term “antigen-binding fragment” of a full length antibody refers toone or more fragments of a full-length antibody that retain the abilityto specifically bind to a target of interest. Examples of bindingfragments encompassed within the term “antigen-binding fragment” of afull length antibody and that retain functionality include (i) a Fabfragment, a monovalent fragment consisting of the VL, VH, CL and CH1domains; (ii) a F(ab′)₂ fragment, a bivalent fragment including two Fabfragments linked by a disulfide bridge at the hinge region; (iii) a Fdfragment consisting of the VH and CH1 domains; (iv) a Fv fragmentconsisting of the VL and VH domains of a single arm of an antibody, (v)a dAb fragment (Ward et al., (1989) Nature 341:544-546), which consistsof a VH domain; and (vi) an isolated complementarity determining region(CDR). Furthermore, although the two domains of the Fv fragment, VL andVH, are coded for by separate genes, they can be joined, usingrecombinant methods, by a synthetic linker that enables them to be madeas a single protein chain in which the VL and VH regions pair to formmonovalent molecules known as single chain Fv (scFv). See e.g., U.S.Pat. Nos. 5,260,203, 4,946,778, and 4,881,175; Bird et al. (1988)Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA85:5879-5883.

Antibody fragments can be obtained using any appropriate techniqueincluding conventional techniques known to those with skill in the art.The term “monospecific antibody” refers to an antibody that displays asingle binding specificity and affinity for a particular target, e.g.,epitope. This term includes a “monoclonal antibody” or “monoclonalantibody composition,” which as used herein refers to a preparation ofantibodies or fragments thereof of single molecular composition,irrespective of how the antibody was generated.

Antibodies are “germlined” by reverting one or more non-germline aminoacids in framework regions to corresponding germline amino acids of theantibody, so long as binding properties are substantially retained.

The inhibition constant (Ki) provides a measure of inhibitor potency; itis the concentration of inhibitor required to reduce enzyme activity byhalf and is not dependent on enzyme or substrate concentrations. Theapparent Ki (K_(i,app)) is obtained at different substrateconcentrations by measuring the inhibitory effect of differentconcentrations of inhibitor (e.g., inhibitory binding protein) on theextent of the reaction (e.g., enzyme activity); fitting the change inpseudo-first order rate constant as a function of inhibitorconcentration to the Morrison equation (Equation 1) yields an estimateof the apparent Ki value. The Ki is obtained from the y-interceptextracted from a linear regression analysis of a plot of Ki,app versussubstrate concentration.

$\begin{matrix}{v = {v_{o} - {v_{o}\left( \frac{\left( {K_{i,{app}} + I + E} \right) - \sqrt{\left( {K_{i,{app}} + I + E} \right)^{2} - {4 \cdot I \cdot E}}}{2 \cdot E} \right)}}} & {{Equation}\mspace{14mu} 1}\end{matrix}$

Where v=measured velocity; v₀=velocity in the absence of inhibitor;K_(i,app)=apparent inhibition constant; I=total inhibitor concentration;and E=total enzyme concentration.

As used herein, “binding affinity” refers to the apparent associationconstant or K_(A). The K_(A) is the reciprocal of the dissociationconstant (K_(D)). A binding protein may, for example, have a bindingaffinity of at least 10⁵, 10⁶, 10⁷, 10⁸, 10⁹, 10¹⁰ and 10¹¹ M⁻¹ for aparticular target molecule, e.g., plasma kallikrein. Higher affinitybinding of a binding protein to a first target relative to a secondtarget can be indicated by a higher K_(A) (or a smaller numerical valueK_(D)) for binding the first target than the K_(A) (or numerical valueK_(D)) for binding the second target. In such cases, the binding proteinhas specificity for the first target (e.g., a protein in a firstconformation or mimic thereof) relative to the second target (e.g., thesame protein in a second conformation or mimic thereof; or a secondprotein). Differences in binding affinity (e.g., for specificity orother comparisons) can be at least 1.5, 2, 3, 4, 5, 10, 15, 20, 37.5,50, 70, 80, 91, 100, 500, 1000, 10,000 or 10⁵ fold.

Binding affinity can be determined by a variety of methods includingequilibrium dialysis, equilibrium binding, gel filtration, ELISA,surface plasmon resonance, or spectroscopy (e.g., using a fluorescenceassay). Exemplary conditions for evaluating binding affinity are inHBS-P buffer (10 mM HEPES pH7.4, 150 mM NaCl, 0.005% (v/v) SurfactantP20). These techniques can be used to measure the concentration of boundand free binding protein as a function of binding protein (or target)concentration. The concentration of bound binding protein ([Bound]) isrelated to the concentration of free binding protein ([Free]) and theconcentration of binding sites for the binding protein on the targetwhere (N) is the number of binding sites per target molecule by thefollowing equation:

[Bound]=N·[Free]/((1/K _(A))+[Free]).

It is not always necessary to make an exact determination of K_(A),though, since sometimes it is sufficient to obtain a quantitativemeasurement of affinity, e.g., determined using a method such as ELISAor FACS analysis, is proportional to K_(A), and thus can be used forcomparisons, such as determining whether a higher affinity is, e.g.,2-fold higher, to obtain a qualitative measurement of affinity, or toobtain an inference of affinity, e.g., by activity in a functionalassay, e.g., an in vitro or in vivo assay.

The term “binding protein” refers to a protein that can interact with atarget molecule. This term is used interchangeably with “ligand.” A“plasma kallikrein binding protein” refers to a protein that caninteract with (e.g., bind) plasma kallikrein, and includes, inparticular, proteins that preferentially or specifically interact withand/or inhibit plasma kallikrein. A protein inhibits plasma kallikreinif it causes a decrease in the activity of plasma kallikrein as comparedto the activity of plasma kallikrein in the absence of the protein andunder the same conditions. In some embodiments, the plasma kallikreinbinding protein is an antibody.

A “conservative amino acid substitution” is one in which the amino acidresidue is replaced with an amino acid residue having a similar sidechain. Families of amino acid residues having similar side chains havebeen defined in the art. These families include amino acids with basicside chains (e.g., lysine, arginine, histidine), acidic side chains(e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g.,glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine),nonpolar side chains (e.g., alanine, valine, leucine, isoleucine,proline, phenylalanine, methionine, tryptophan), beta-branched sidechains (e.g., threonine, valine, isoleucine) and aromatic side chains(e.g., tyrosine, phenylalanine, tryptophan, histidine).

It is possible for one or more framework and/or CDR amino acid residuesof a binding protein to include one or more mutations (e.g.,substitutions (e.g., conservative substitutions or substitutions ofnon-essential amino acids), insertions, or deletions) relative to abinding protein described herein. A plasma kallikrein binding proteinmay have mutations (e.g., substitutions (e.g., conservativesubstitutions or substitutions of non-essential amino acids),insertions, or deletions) (e.g., at least one, two, three, or four,and/or less than 15, 12, 10, 9, 8, 7, 6, 5, 4, 3, or 2 mutations)relative to a binding protein described herein, e.g., mutations which donot have a substantial effect on protein function. The mutations can bepresent in framework regions, CDRs, and/or constant regions. In someembodiments, the mutations are present in a framework region. In someembodiments, the mutations are present in a CDR. In some embodiments,the mutations are present in a constant region. Whether or not aparticular substitution will be tolerated, i.e., will not adverselyaffect biological properties, such as binding activity, can bepredicted, e.g., by evaluating whether the mutation is conservative orby the method of Bowie, et al. (1990) Science 247:1306-1310.

Motif sequences for biopolymers can include positions which can bevaried amino acids. For example, the symbol “X” in such a contextgenerally refers to any amino acid (e.g., any of the twenty naturalamino acids) unless otherwise specified, e.g., to refer to anynon-cysteine amino acid. Other allowed amino acids can also be indicatedfor example, using parentheses and slashes. For example, “(A/W/F/N/Q)”means that alanine, tryptophan, phenylalanine, asparagine, and glutamineare allowed at that particular position.

An “effectively human” immunoglobulin variable region is animmunoglobulin variable region that includes a sufficient number ofhuman framework amino acid positions such that the immunoglobulinvariable region does not elicit an immunogenic response in a normalhuman. An “effectively human” antibody is an antibody that includes asufficient number of human amino acid positions such that the antibodydoes not elicit an immunogenic response in a normal human.

An “epitope” refers to the site on a target compound that is bound by abinding protein (e.g., an antibody such as a Fab or full lengthantibody). In the case where the target compound is a protein, the sitecan be entirely composed of amino acid components, entirely composed ofchemical modifications of amino acids of the protein (e.g., glycosylmoieties), or composed of combinations thereof. Overlapping epitopesinclude at least one common amino acid residue, glycosyl group,phosphate group, sulfate group, or other molecular feature.

A first binding protein (e.g., antibody) “binds to the same epitope” asa second binding protein (e.g., antibody) if the first binding proteinbinds to the same site on a target compound that the second bindingprotein binds, or binds to a site that overlaps (e.g., 50%, 60%, 70%,80%, 90%, or 100% overlap, e.g., in terms of amino acid sequence orother molecular feature (e.g., glycosyl group, phosphate group, orsulfate group)) with the site that the second binding protein binds.

A first binding protein (e.g., antibody) “competes for binding” with asecond binding protein (e.g., antibody) if the binding of the firstbinding protein to its epitope decreases (e.g., by 10%, 20%, 30%, 40%,50%, 60%, 70%, 80%, 90%, 100%, or more) the amount of the second bindingprotein that binds to its epitope. The competition can be direct (e.g.,the first binding protein binds to an epitope that is the same as, oroverlaps with, the epitope bound by the second binding protein), orindirect (e.g., the binding of the first binding protein to its epitopecauses a steric change in the target compound that decreases the abilityof the second binding protein to bind to its epitope).

Calculations of “homology” or “sequence identity” between two sequences(the terms are used interchangeably herein) are performed as follows.The sequences are aligned for optimal comparison purposes (e.g., gapscan be introduced in one or both of a first and a second amino acid ornucleic acid sequence for optimal alignment and non-homologous sequencescan be disregarded for comparison purposes). The optimal alignment isdetermined as the best score using the GAP program in the GCG softwarepackage with a Blossum 62 scoring matrix with a gap penalty of 12, a gapextend penalty of 4, and a frameshift gap penalty of 5. The amino acidresidues or nucleotides at corresponding amino acid positions ornucleotide positions are then compared. When a position in the firstsequence is occupied by the same amino acid residue or nucleotide as thecorresponding position in the second sequence, then the molecules areidentical at that position (as used herein amino acid or nucleic acid“identity” is equivalent to amino acid or nucleic acid “homology”). Thepercent identity between the two sequences is a function of the numberof identical positions shared by the sequences.

In a preferred embodiment, the length of a reference sequence alignedfor comparison purposes is at least 30%, preferably at least 40%, morepreferably at least 50%, even more preferably at least 60%, and evenmore preferably at least 70%, 80%, 90%, 92%, 95%, 97%, 98%, or 100% ofthe length of the reference sequence. For example, the referencesequence may be the length of the immunoglobulin variable domainsequence.

A “humanized” immunoglobulin variable region is an immunoglobulinvariable region that is modified to include a sufficient number of humanframework amino acid positions such that the immunoglobulin variableregion does not elicit an immunogenic response in a normal human.Descriptions of “humanized” immunoglobulins include, for example, U.S.Pat. No. 6,407,213 and U.S. Pat. No. 5,693,762.

As used herein, the term “hybridizes under low stringency, mediumstringency, high stringency, or very high stringency conditions”describes conditions for hybridization and washing. Guidance forperforming hybridization reactions can be found in Current Protocols inMolecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. Aqueousand nonaqueous methods are described in that reference and either can beused. Specific hybridization conditions referred to herein are asfollows: (1) low stringency hybridization conditions in 6× sodiumchloride/sodium citrate (SSC) at about 45° C., followed by two washes in0.2×SSC, 0.1% SDS at least at 50° C. (the temperature of the washes canbe increased to 55° C. for low stringency conditions); (2) mediumstringency hybridization conditions in 6×SSC at about 45° C., followedby one or more washes in 0.2×SSC, 0.1% SDS at 60° C.; (3) highstringency hybridization conditions in 6×SSC at about 45° C., followedby one or more washes in 0.2×SSC, 0.1% SDS at 65° C.; and (4) very highstringency hybridization conditions are 0.5M sodium phosphate, 7% SDS at65° C., followed by one or more washes at 0.2×SSC, 1% SDS at 65° C. Veryhigh stringency conditions (4) are the preferred conditions and the onesthat should be used unless otherwise specified. The disclosure includesnucleic acids that hybridize with low, medium, high, or very highstringency to a nucleic acid described herein or to a complementthereof, e.g., nucleic acids encoding a binding protein describedherein. The nucleic acids can be the same length or within 30, 20, or10% of the length of the reference nucleic acid. The nucleic acid cancorrespond to a region encoding an immunoglobulin variable domainsequence described herein.

An “isolated composition” refers to a composition that is removed fromat least 90% of at least one component of a natural sample from whichthe isolated composition can be obtained. Compositions producedartificially or naturally can be “compositions of at least” a certaindegree of purity if the species or population of species of interest isat least 5, 10, 25, 50, 75, 80, 90, 92, 95, 98, or 99% pure on aweight-weight basis.

An “isolated” protein refers to a protein that is removed from at least90% of at least one component of a natural sample from which theisolated protein can be obtained. Proteins can be “of at least” acertain degree of purity if the species or population of species ofinterest is at least 5, 10, 25, 50, 75, 80, 90, 92, 95, 98, or 99% pureon a weight-weight basis.

The term “modulator” refers to a polypeptide, nucleic acid,macromolecule, complex, molecule, small molecule, compound, species orthe like (naturally-occurring or non-naturally-occurring), or an extractmade from biological materials such as bacteria, plants, fungi, oranimal cells or tissues, that may be capable of causing modulation.Modulators may be evaluated for potential activity as inhibitors oractivators (directly or indirectly) of a functional property, biologicalactivity or process, or combination of them, (e.g., agonist, partialantagonist, partial agonist, inverse agonist, antagonist, anti-microbialagents, inhibitors of microbial infection or proliferation, and thelike) by inclusion in assays. In such assays, many modulators may bescreened at one time. The activity of a modulator may be known, unknownor partially known.

A “non-essential” amino acid residue is a residue that can be alteredfrom the wild-type sequence of the binding agent, e.g., the antibody,without abolishing or more preferably, without substantially altering abiological activity, whereas changing an “essential” amino acid residueresults in a substantial loss of activity.

A “patient,” “subject” or “host” (these terms are used interchangeably)to be treated by the subject method may mean either a human or non-humananimal.

The terms “prekallikrein” and “preplasma kallikrein” are usedinterchangeably herein and refer to the zymogen form of active plasmakallikrein, which is also known as prekallikrein.

The term “preventing” or to “prevent” a disease in a subject refers tosubjecting the subject to a pharmaceutical treatment, e.g., theadministration of a drug, such that at least one symptom of the diseaseis prevented, that is, administered prior to clinical manifestation ofthe unwanted condition (e.g., disease or other unwanted state of thehost animal) so that it protects the host against developing theunwanted condition. “Preventing” a disease may also be referred to as“prophylaxis” or “prophylactic treatment.”

As used herein, the term “substantially identical” (or “substantiallyhomologous”) is used herein to refer to a first amino acid or nucleicacid sequence that contains a sufficient number of identical orequivalent (e.g., with a similar side chain, e.g., conserved amino acidsubstitutions) amino acid residues or nucleotides to a second amino acidor nucleic acid sequence such that the first and second amino acid ornucleic acid sequences have (or encode proteins having) similaractivities, e.g., a binding activity, a binding preference, or abiological activity. In the case of antibodies, the second antibody hasthe same specificity and has at least 50%, at least 25%, or at least 10%of the affinity relative to the same antigen.

Sequences similar or homologous (e.g., at least about 85% sequenceidentity) to the sequences disclosed herein are also part of thisapplication. In some embodiments, the sequence identity can be about85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher. In someembodiments, a plasma kallikrein binding protein can have about 85%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequenceidentity to a binding protein described herein. In some embodiments, aplasma kallikrein binding protein can have about 85%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity in the HCand/or LC framework regions (e.g., HC and/or LC FR 1, 2, 3, and/or 4) toa binding protein described herein. In some embodiments, a plasmakallikrein binding protein can have about 85%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99% or higher sequence identity in the HC and/or LCCDRs (e.g., HC and/or LC CDR1, 2, and/or 3) to a binding proteindescribed herein. In some embodiments, a plasma kallikrein bindingprotein can have about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or higher sequence identity in the constant region (e.g., CH1, CH2,CH3, and/or CL1) to a binding protein described herein.

In addition, substantial identity exists when the nucleic acid segmentshybridize under selective hybridization conditions (e.g., highlystringent hybridization conditions), to the complement of the strand.The nucleic acids may be present in whole cells, in a cell lysate, or ina partially purified or substantially pure form.

Statistical significance can be determined by any art known method.Exemplary statistical tests include: the Students T-test, Mann Whitney Unon-parametric test, and Wilcoxon non-parametric statistical test. Somestatistically significant relationships have a P value of less than 0.05or 0.02. Particular binding proteins may show a difference, e.g., inspecificity or binding that are statistically significant (e.g., Pvalue<0.05 or 0.02). The terms “induce”, “inhibit”, “potentiate”,“elevate”, “increase”, “decrease” or the like, e.g., which denotedistinguishable qualitative or quantitative differences between twostates, may refer to a difference, e.g., a statistically significantdifference, between the two states.

A “therapeutically effective dosage” preferably modulates a measurableparameter, e.g., plasma kallikrein activity, by a statisticallysignificant degree or at least about 20%, more preferably by at leastabout 40%, even more preferably by at least about 60%, and still morepreferably by at least about 80% relative to untreated subjects. Theability of a compound to modulate a measurable parameter, e.g., adisease-associated parameter, can be evaluated in an animal model systempredictive of efficacy in human disorders and conditions, e.g.,rheumatoid arthritis or oral mucositis. Alternatively, this property ofa composition can be evaluated by examining the ability of the compoundto modulate a parameter in vitro.

“Treating” a disease (or condition) in a subject or “treating” a subjecthaving a disease refers to subjecting the subject to a pharmaceuticaltreatment, e.g., the administration of a drug, such that at least onesymptom of the disease is cured, alleviated or decreased.

The term “preventing” a disease in a subject refers to subjecting thesubject to a pharmaceutical treatment, e.g., the administration of adrug, such that at least one symptom of the disease is prevented, thatis, administered prior to clinical manifestation of the unwantedcondition (e.g., disease or other unwanted state of the host animal) sothat it protects the host against developing the unwanted condition.“Preventing” a disease may also be referred to as “prophylaxis” or“prophylactic treatment.”

A “prophylactically effective amount” refers to an amount effective, atdosages and for periods of time necessary, to achieve the desiredprophylactic result. Typically, because a prophylactic dose is used insubjects prior to or at an earlier stage of disease, theprophylactically effective amount will be less than the therapeuticallyeffective amount.

As used herein the term “DX-2922” as used inchangeably with the term“X101-A01”. Other variants of this antibody are described below.

Antibody Identification Description X63-G06 Non-germlined Fab discoveredusing ROLIC, same HC but different LC as M160-G12 X81-B01 Germlined IgGproduced in HEK 293T cells X101-A01 Germlined IgG produced in CHO cells,same HC and LC sequence as X81-B01 DX-2922 Alternate nomenclature forX101-A01

Plasma Kallikrein Binding Proteins

Plasma kallikrein binding proteins can be full-length (e.g., an IgG(e.g., an IgG1, IgG2, IgG3, IgG4), IgM, IgA (e.g., IgA1, IgA2), IgD, andIgE) or can include only an antigen-binding fragment (e.g., a Fab,F(ab′)2 or scFv fragment. The binding protein can include two heavychain immunoglobulins and two light chain immunoglobulins, or can be asingle chain antibody. Plasma kallikrein binding proteins can berecombinant proteins such as humanized, CDR grafted, chimeric,deimmunized, or in vitro generated antibodies, and may optionallyinclude constant regions derived from human germline immunoglobulinsequences. In one embodiment, the plasma kallikrein binding protein is amonoclonal antibody.

In one aspect, the disclosure features a protein (e.g., an isolatedprotein) that binds to plasma kallikrein (e.g., human plasma kallikreinand/or murine kallikrein) and includes at least one immunoglobulinvariable region. For example, the protein includes a heavy chain (HC)immunoglobulin variable domain sequence and/or a light chain (LC)immunoglobulin variable domain sequence. In one embodiment, the proteinbinds to and inhibits plasma kallikrein, e.g., human plasma kallikreinand/or murine kallikrein.

The protein can include one or more of the following characteristics:(a) a human CDR or human framework region; (b) the HC immunoglobulinvariable domain sequence comprises one or more (e.g., 1, 2, or 3) CDRsthat are at least 85, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or100% identical to a CDR of a HC variable domain described herein; (c)the LC immunoglobulin variable domain sequence comprises one or more(e.g., 1, 2, or 3) CDRs that are at least 85, 88, 89, 90, 91, 92, 93,94, 95, 96, 97, 98, 99, or 100% identical to a CDR of a LC variabledomain described herein; (d) the LC immunoglobulin variable domainsequence is at least 85, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99,or 100% identical to a LC variable domain described herein (e.g.,overall or in framework regions or CDRs); (e) the HC immunoglobulinvariable domain sequence is at least 85, 88, 89, 90, 91, 92, 93, 94, 95,96, 97, 98, 99, or 100% identical to a HC variable domain describedherein (e.g., overall or in framework regions or CDRs); (f) the proteinbinds an epitope bound by a protein described herein, or competes forbinding with a protein described herein; (g) a primate CDR or primateframework region; (h) the HC immunoglobulin variable domain sequencecomprises a CDR1 that differs by at least one amino acid but by no morethan 2 or 3 amino acids from the CDR1 of a HC variable domain describedherein; (i) the HC immunoglobulin variable domain sequence comprises aCDR2 that differs by at least one amino acid but by no more than 2, 3,4, 5, 6, 7, or 8 amino acids from the CDR2 of a HC variable domaindescribed herein; (j) the HC immunoglobulin variable domain sequencecomprises a CDR3 that differs by at least one amino acid but by no morethan 2, 3, 4, 5, or 6 amino acids from the CDR3 of a HC variable domaindescribed herein; (k) the LC immunoglobulin variable domain sequencecomprises a CDR1 that differs by at least one amino acid but by no morethan 2, 3, 4, or 5 amino acids from the CDR1 of a LC variable domaindescribed herein; (1) the LC immunoglobulin variable domain sequencecomprises a CDR2 that differs by at least one amino acid but by no morethan 2, 3, or 4 amino acids from the CDR2 of a LC variable domaindescribed herein; (m) the LC immunoglobulin variable domain sequencecomprises a CDR3 that differs by at least one amino acid but by no morethan 2, 3, 4, or 5 amino acids from the CDR3 of a LC variable domaindescribed herein; (n) the LC immunoglobulin variable domain sequencediffers by at least one amino acid but by no more than 2, 3, 4, 5, 6, 7,8, 9, or 10 amino acids from a LC variable domain described herein(e.g., overall or in framework regions or CDRs); and (o) the HCimmunoglobulin variable domain sequence differs by at least one aminoacid but by no more than 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids froma HC variable domain described herein (e.g., overall or in frameworkregions or CDRs).

The plasma kallikrein binding protein may be an isolated protein (e.g.,at least 70, 80, 90, 95, or 99% free of other proteins). In someembodiments, the plasma kallikrein binding protein, or compositionthereof, is isolated from antibody cleavage fragments (e.g., cleavedDX-2922) that are inactive or partially active (e.g., bind plasmakallikrein with a Ki, app of 5000 nM or greater) compared to the plasmakallikrein binding protein. For example, the plasma kallikrein bindingprotein is at least 70% free of such antibody cleavage fragments; inother embodiments the binding protein is at least 80%, at least 90%, atleast 95%, at least 99% or even 100% free from antibody cleavagefragments that are inactive or partially active.

The plasma kallikrein binding protein may additionally inhibit plasmakallikrein, e.g., human plasma kallikrein.

In some embodiments, the plasma kallikrein binding protein does not bindprekallikrein (e.g., human prekallikrein and/or murine prekallikrein),but binds to the active form of plasma kallikrein (e.g., human plasmakallikrein and/or murine kallikrein).

In certain embodiments, the protein binds at or near the active site ofthe catalytic domain of plasma kallikrein, or a fragment thereof, orbinds an epitope that overlaps with the active site of plasmakallikrein.

In some aspects, the protein binds the same epitope or competes forbinding with a protein described herein.

In some embodiments, the protein competes with or binds the same epitopeas M162-A04, M160-G12, M142-H08, X63-G06, X101-A01, X81-B01, X67-D03,X67-G04, X115-B07, X115-D05, X115-E09, X115-H06, X115-A03, X115-D01,X115-F02, X115-G04, M29-D09, M145-D11, M06-D09 and M35-G04.

In some embodiments, the protein binds to (e.g., positions on plasmakallikrein corresponding to) CLIPS peptide C1, C2, C3, C4, C5, C6, orC7, or more than one of these peptides, e.g., the protein binds to C5and C6. CLIPS peptides C1-C7 are peptides in plasma kallikreinidentified by CLIPS epitope mapping (see FIGS. 9 and 10A-10C). C1corresponds to positions 55-67 of the catalytic domain, C2 to positions81-94, C3 to positions 101-108, C4 to positions 137-151, C5 to positions162-178, C6 to positions 186-197, and C7 to positions 214-217 of plasmakallikrein.

In some embodiments, the protein binds to an epitope shown in FIG. 9.

In some embodiments, the protein binds to one or more amino acids thatform the catalytic triad of plasma kallikrein: His434, Asp483, and/orSer578 (numbering based on the human sequence).

In some embodiments, the protein binds one or more amino acids of:Arg551, Gln553, Tyr555, Thr558, and/or Arg560 (numbering based on thehuman sequence). In some embodiments, the plasma kallikrein bindingprotein binds one or more amino acids of: S478, N481, S525, and K526(numbering based on the human kallikrein sequence).

In some embodiments, the protein binds to one or more amino acids ofSer479, Tyr563, and/or Asp585 (numbering based on the human sequence).

The active site cleft of plasma kallikrein contains three amino acidsthat form the catalytic triad (His434, Asp483, and Ser578) and result inenzymatic hydrolysis of bound substrate (catalytic triad residues areunderlined in FIG. 10). The peptides selected for the CLIPS epitopemapping analysis were determined to be surface accessible and eitherform or surround the vicinity of the active site. Peptide C1 containsthe active site histidine 434. Peptide C3 contains the active siteaspartate 483. Peptide C6 contains the active site serine 578. It ispossible for an antibody to bind multiple surface exposed amino acidsthat are discontinuous in amino acid sequence. For example, by CLIPsanalysis, X81-B01 appears to bind the C2, C3, C5 and the C6 peptides.

In some embodiments, the protein binds to an epitope that includes oneor more amino acids from CLIPS peptide C1, peptide C2, peptide C3,peptide C4, peptide C5, peptide C6, or peptide C7.

In some embodiments, the protein binds to an epitope that includes aminoacids from at least 2 different CLIPS peptides, e.g., from at least twoof peptide C1, peptide C2, peptide C3, peptide C4, peptide C5, peptideC6, or peptide C7.

The protein can bind to plasma kallikrein, e.g., human plasmakallikrein, with a binding affinity of at least 10⁵, 10⁶, 10⁷, 10⁸, 10⁹,10¹⁰ and 10¹¹ M⁻¹. In one embodiment, the protein binds to human plasmakallikrein with a K_(off) slower than 1×10⁻³, 5×10⁻⁴ s⁻¹, or 1×10⁻⁴ s⁻¹.In one embodiment, the protein binds to human plasma kallikrein with aK_(on) faster than 1×10², 1×10³, or 5×10³ M⁻¹s⁻¹. In one embodiment, theprotein binds to plasma kallikrein, but does not bind to tissuekallikrein and/or plasma prekallikrein (e.g., the protein binds totissue kallikrein and/or plasma prekallikrein less effectively (e.g.,5-, 10-, 50-, 100-, or 1000-fold less or not at all, e.g., as comparedto a negative control) than it binds to plasma kallikrein.

In one embodiment, the protein inhibits human plasma kallikreinactivity, e.g., with a Ki of less than 10⁻⁵, 10⁻⁶, 10⁻⁷, 10⁻⁸, 10⁻⁹, and10⁻¹⁰ M. The protein can have, for example, an IC₅₀ of less than 100 nM,10 nM, 1, 0.5, or 0.2 nM. For example, the protein may modulate plasmakallikrein activity, as well as the production of Factor XIIa (e.g.,from Factor XII) and/or bradykinin (e.g., from high-molecular-weightkininogen (HMWK)). The protein may inhibit plasma kallikrein activity,and/or the production of Factor XIIa (e.g., from Factor XII) and/orbradykinin (e.g., from high-molecular-weight kininogen (HMWK)). Theaffinity of the protein for human plasma kallikrein can be characterizedby a K_(D) of less than 100 nm, less than 10 nM, less than 5 nM, lessthan 1 nM, less than 0.5 nM. In one embodiment, the protein inhibitsplasma kallikrein, but does not inhibit tissue kallikrein (e.g., theprotein inhibits tissue kallikrein less effectively (e.g., 5-, 10-, 50-,100-, or 1000-fold less or not at all, e.g., as compared to a negativecontrol) than it inhibits plasma kallikrein.

In some embodiments, the protein has an apparent inhibition constant(K_(i,app)) of less than 1000, 500, 100, 5, 1, 0.5 or 0.2 nM.

Plasma kallikrein binding proteins may be antibodies. Plasma kallikreinbinding antibodies may have their HC and LC variable domain sequencesincluded in a single polypeptide (e.g., scFv), or on differentpolypeptides (e.g., IgG or Fab).

In a preferred embodiment, the protein is an antibody (e.g., a humanantibody) having the light and heavy chains of antibodies selected fromthe group consisting of M162-A04, M160-G12, M142-H08 X63-G06, X101-A01,X81-B01, X67-D03, X67-G04, DX-2922, X115-B07, X115-D05, X115-E09,X115-H06, X115-A03, X115-D01, X115-F02, X115-G04, M29-D09, M145-D11,M06-D09 and M35-G04.

In a preferred embodiment, the protein is an antibody (e.g., a humanantibody) having the heavy chain of an antibody selected from the groupconsisting of: M162-A04, M160-G12, M142-H08, X63-G06, X101-A01, X81-B01,X67-D03, X67-G04, X115-B07, X115-D05, X115-E09, X115-H06, X115-A03,X115-D01, X115-F02, X115-G04, M29-D09, M145-D11, M06-D09 and M35-G04.

In a preferred embodiment, the protein is an antibody (e.g., a humanantibody) having the light chain of an antibody selected from the groupconsisting of: M162-A04, M160-G12, M142-H08 X63-G06, X101-A01, X81-B01,X67-D03, X67-G04, X115-B07, X115-D05, X115-E09, X115-H06, X115-A03,X115-D01, X115-F02, X115-G04, M29-D09, M145-D11, M06-D09 and M35-G04.

In a preferred embodiment, the protein is an antibody (e.g., a humanantibody) having light and heavy antibody variable regions of anantibody selected from the group consisting of M162-A04, M160-G12,M142-H08, X63-G06, X101-A01, X81-B01, X67-D03, X67-G04, X115-B07,X115-D05, X115-E09, X115-H06, X115-A03, X115-D01, X115-F02, X115-G04,M29-D09, M145-D11, M06-D09 and M35-G04.

In a preferred embodiment, the protein is an antibody (e.g., a humanantibody) having a heavy chain antibody variable region of an antibodyselected from the group consisting of: M162-A04, M160-G12, M142-H08,X63-G06, X101-A01, X81-B01, X67-D03, X67-G04, X115-B07, X115-D05,X115-E09, X115-H06, X115-A03, X115-D01, X115-F02, X115-G04, M29-D09,M145-D11, M06-D09 and M35-G04.

In a preferred embodiment, the protein is an antibody (e.g., a humanantibody) having a light chain antibody variable region of an antibodyselected from the group consisting of: M162-A04, M160-G12, M142-H08,X63-G06, X101-A01, X81-B01, X67-D03, X67-G04, X115-B07, X115-D05,X115-E09, X115-H06, X115-A03, X115-D01, X115-F02, X115-G04, M29-D09,M145-D11, M06-D09 and M35-G04.

In a preferred embodiment, the protein is an antibody (e.g., a humanantibody) having one or more (e.g., 1, 2, or 3) heavy chain CDRsselected from the corresponding CDRs of the group of heavy chainsconsisting of M162-A04, M160-G12, M142-H08, X63-G06, X101-A01, X81-B01,X67-D03, X67-G04, X115-B07, X115-D05, X115-E09, X115-H06, X115-A03,X115-D01, X115-F02, X115-G04, M29-D09, M145-D11, M06-D09 and M35-G04.

In a preferred embodiment, the protein is an antibody (e.g., a humanantibody) having one or more (e.g., 1, 2, or 3) light chain CDRsselected from the corresponding CDRs of the group of light chainsconsisting of M162-A04, M160-G12, M142-H08, X63-G06, X101-A01, X81-B01,X67-D03, X67-G04, X115-B07, X115-D05, X115-E09, X115-H06, X115-A03,X115-D01, X115-F02, X115-G04, M29-D09, M145-D11, M06-D09 and M35-G04.

In a preferred embodiment, the protein is an antibody (e.g., a humanantibody) having one or more (e.g., 1, 2, or 3) heavy chain CDRs and oneor more (e.g., 1, 2, or 3) light chain CDRs selected from thecorresponding CDRs of the group of light chains consisting of M162-A04,M160-G12, M142-H08, X63-G06, X101-A01, X81-B01, X67-D03, X67-G04,X115-B07, X115-D05, X115-E09, X115-H06, X115-A03, X115-D01, X115-F02,X115-G04, M29-D09, M145-D11, M06-D09 and M35-G04.

In one embodiment, the HC and LC variable domain sequences arecomponents of the same polypeptide chain. In another, the HC and LCvariable domain sequences are components of different polypeptidechains. For example, the protein is an IgG, e.g., IgG1, IgG2, IgG3, orIgG4. The protein can be a soluble Fab. In other implementations theprotein includes a Fab2′, scFv, minibody, scFv::Fc fusion, Fab::HSAfusion, HSA::Fab fusion, Fab::HSA::Fab fusion, or other molecule thatcomprises the antigen combining site of one of the binding proteinsherein. The VH and VL regions of these Fabs can be provided as IgG, Fab,Fab2, Fab2′, scFv, PEGylated Fab, PEGylated scFv, PEGylated Fab2,VH::CH1::HSA+LC, HSA::VH::CH1+LC, LC::HSA+VH::CH1, HSA::LC+VH::CH1, orother appropriate construction.

In one embodiment, the protein is a human or humanized antibody or isnon-immunogenic in a human. For example, the protein includes one ormore human antibody framework regions, e.g., all human frameworkregions, or framework regions at least 85, 88, 89, 90, 91, 92, 93, 94,95, 96, 97, 98, 99% identical to human framework regions. In oneembodiment, the protein includes a human Fc domain, or an Fc domain thatis at least 95, 96, 97, 98, or 99% identical to a human Fc domain.

In one embodiment, the protein is a primate or primatized antibody or isnon-immunogenic in a human. For example, the protein includes one ormore primate antibody framework regions, e.g., all primate frameworkregions, or framework regions at least 85, 88, 89, 90, 91, 92, 93, 94,95, 96, 97, 98, 99% identical to primate framework regions. In oneembodiment, the protein includes a primate Fc domain, or an Fc domainthat is at least 95, 96, 97, 98, or 99% identical to a primate Fcdomain. “Primate” includes humans (Homo sapiens), chimpanzees (Pantroglodytes and Pan paniscus (bonobos)), gorillas (Gorilla gorilla),gibons, monkeys, lemurs, aye-ayes (Daubentonia madagascariensis), andtarsiers.

In some embodiments, the affinity of the primate antibody for humanplasma kallikrein is characterized by a K_(D) of less than 1000, 500,100, 10, 5, 1, 0.5 nM, e.g., less than 10 nM, less than 1 nM, or lessthan 0.5 nM.

In certain embodiments, the protein includes no sequences from mice orrabbits (e.g., is not a murine or rabbit antibody).

In some aspects, the disclosure provides the use of proteins (e.g.,binding proteins, e.g., antibodies) (e.g., the proteins describedherein) that bind to plasma kallikrein (e.g., human plasma kallikrein)and include at least one immunoglobin variable region in methods fortreating (or preventing) a plasma kallikrein associated disorder orcondition. For example, the plasma kallikrein binding protein includes aheavy chain (HC) immunoglobulin variable domain sequence and a lightchain (LC) immunoglobulin variable domain sequence. A number ofexemplary plasma kallikrein binding proteins are described herein.

The plasma kallikrein binding protein may be an isolated protein (e.g.,at least 70, 80, 90, 95, or 99% free of other proteins).

The plasma kallikrein binding protein may additionally inhibit plasmakallikrein, e.g., human plasma kallikrein and/or murine plasmakallikrein. In some embodiments, it may be preferred to have a plasmakallikrein binding protein bind to both human and murine plasmakallikrein, as these antibodies can be tested for efficacy in a mousemodel.

Plasma Kallikrein

Exemplary plasma kallikrein sequences against which plasma kallikreinbinding proteins may be developed can include human, mouse, or ratplasma kallikrein amino acid sequences, a sequence that is 80%, 85%,90%, 95%, 96%, 97%, 98%, or 99% identical to one of these sequences, ora fragment thereof, e.g., of a sequence provided below.

The sequence of human plasma kallikrein that was used in selections andsubsequent screening is shown below (accession number NP_000883.2). Thehuman plasma kallikrein (86 kDa) that was used was purified from humanplasma and activated with factor XIIa by a commercial vendor. FactorXIIa activates prekallikrein by cleaving the polypeptide sequence at asingle site (between Arg371-Ile372, cleavage site marked by “/” in thesequence below) to generate active plasma kallikrein, which thenconsists of two disulfide linked polypeptides; a heavy chain ofapproximately 52 kDa and a catalytic domain of approximately 34 kDa[Colman and Schmaier, (1997) “Contact System: A Vascular BiologyModulator With Anticoagulant, Profibrinolytic, Antiadhesive, andProinflammatory Attributes” Blood, 90, 3819-3843]

(SEQ ID NO: 1) GCLTQLYENAFFRGGDVASMYTPNAQYCQMRCTFHPRCLLFSFLPASSINDMEKRFGCFLKDSVTGTLPKVHRTGAVSGHSLKQCGHQISACHRDIYKGVDMRGVNFNVSKVSSVEECQKRCTSNIRCQFFSYATQTFHKAEYRNNCLLKYSPGGTPTAIKVLSNVESGFSLKPCALSEIGCHMNIFQHLAFSDVDVARVLTPDAFVCRTICTYHPNCLFFTFYTNVWKIESQRNVCLLKTSESGTPSSSTPQENTISGYSLLTCKRTLPEPCHSKIYPGVDFGGEELNVTFVKGVNVCQETCTKMIRCQFFTYSLLPEDCKEEKCKCFLRLSMDGSPTRIAYGTQGSSGYSLRLCNTGDNSVCTTKTSTR/IVGGTNSSWGEWPWQVSLQVKLTAQRHLCGGSLIGHQWVLTAAHCFDGLPLQDVWRIYSGILNLSDITKDTPFSQIKEIIIHQNYKVSEGNHDIALIKLQAPLNYTEFQKPICLPSKGDTSTIYTNCWVTGWGFSKEKGEIQNILQKVNIPLVTNEECQKRYQDYKITQRMVCAGYKEGGKDACKGDSGGPLVCKHNGMWRLVGITSWGEGCARREQPGVYTKVAEYM DWILEKTQSSDGKAQMQSPA

The human, mouse, and rat prekallikrein amino acid sequences, and themRNA sequences encoding the same, are illustrated below. The sequencesof prekallikrein are the same as plasma kallikrein, except that activeplasma kallikrein (pkal) has the single polypeptide chain cleaved at asingle position (indicated by the “/”) to generate two chains. Thesequences provided below are full sequences that include signalsequences. On secretion from the expressing cell, it is expected thatthe signal sequences are removed.

Human plasma kallikrein (ACCESSION: NP_000883.2) >gi|78191798|ref|NP_000883.2|plasma kallikrein B1 precursor [Homo sapiens] (SEQ ID NO: 2)MILFKQATYFISLFATVSCGCLTQLYENAFFRGGDVASMYTPNAQYCQMRCTFHPRCLLFSFLPASSINDMEKREGCFLKDSVTGTLPKVHRTGAVSGHSLKQCGHQISACHRDIYKGVDMRGVNFNVSKVSSVEECQKRCTSNIRCQFFSYATQTFHKAEYRNNCLLKYSPGGTPTAIKVLSNVESGESLKPCALSEIGCHMNIFQHLAFSDVDVARVLTPDAFVCRTICTYHPNCLFFTFYTNVWKIESQRNVCLLKTSESGTPSSSTPQENTISGYSLLTCKRTLPEPCHSKIYPGVDEGGEELNVTFVKGVNVCQETCTKMIRCQFFTYSLLPEDCKEEKCKCFLRLSMDGSPTRIAYGTQGSSGYSLRLCNTGDNSVCTTKTSTRIVGGTNSSWGEWPWQVSLQVKLTAQRHLCGGSLIGHQWVLTAAHCFDGLPLQDVWRIYSGILNLSDITKDTPFSQIKEIIIHQNYKVSEGNHDIALIKLQAPLNYTEFQKPICLPSKGDTSTIYTNCWVTGWGESKEKGEIQNILQKVNIPLVTNEECQKRYQDYKITQRMVCAGYKEGGKDACKGDSGGPLVCKHNGMWRLVGITSWGEGCARREQPGVYTKVAEYMDWILEKTQSSDGKAQMQSPAHuman plasma kallikrein mRNA (ACCESSION: NM_000892) >gi|78191797|ref|NM_000892.3|Homo sapiens kallikrein B, plasma (Fletcher factor) 1 (KLKB1), mRNA(SEQ ID NO: 3)AGAACAGCTTGAAGACCGTTCATTTTTAAGTGACAAGAGACTCACCTCCAAGAAGCAATTGTGTTTTCAGAATGATTTTATTCAAGCAAGCAACTTATTTCATTTCCTTGTTTGCTACAGTTTCCTGTGGATGTCTGACTCAACTCTATGAAAACGCCTTCTTCAGAGGTGGGGATGTAGCTTCCATGTACACCCCAAATGCCCAATACTGCCAGATGAGGTGCACATTCCACCCAAGGTGTTTGCTATTCAGTTTTCTTCCAGCAAGTTCAATCAATGACATGGAGAAAAGGTTTGGTTGCTTCTTGAAAGATAGTGTTACAGGAACCCTGCCAAAAGTACATCGAACAGGTGCAGTTTCTGGACATTCCTTGAAGCAATGTGGTCATCAAATAAGTGCTTGCCATCGAGACATTTATAAAGGAGTTGATATGAGAGGAGTCAATTTTAATGTGTCTAAGGTTAGCAGTGTTGAAGAATGCCAAAAAAGGTGCACCAGTAACATTCGCTGCCAGTTTTTTTCATATGCCACGCAAACATTTCACAAGGCAGAGTACCGGAACAATTGCCTATTAAAGTACAGTCCCGGAGGAACACCTACCGCTATAAAGGTGCTGAGTAACGTGGAATCTGGATTCTCACTGAAGCCCTGTGCCCTTTCAGAAATTGGTTGCCACATGAACATCTTCCAGCATCTTGCGTTCTCAGATGTGGATGTTGCCAGGGTTCTCACTCCAGATGCTTTTGTGTGTCGGACCATCTGCACCTATCACCCCAACTGCCTCTTCTTTACATTCTATACAAATGTATGGAAAATCGAGTCACAAAGAAATGTTTGTCTTCTTAAAACATCTGAAAGTGGCACACCAAGTTCCTCTACTCCTCAAGAAAACACCATATCTGGATATAGCCTTTTAACCTGCAAAAGAACTTTACCTGAACCCTGCCATTCTAAAATTTACCCGGGAGTTGACTTTGGAGGAGAAGAATTGAATGTGACTTTTGTTAAAGGAGTGAATGTTTGCCAAGAGACTTGCACAAAGATGATTCGCTGTCAGTTTTTCACTTATTCTTTACTCCCAGAAGACTGTAAGGAAGAGAAGTGTAAGTGTTTCTTAAGATTATCTATGGATGGTTCTCCAACTAGGATTGCGTATGGGACACAAGGGAGCTCTGGTTACTCTTTGAGATTGTGTAACACTGGGGACAACTCTGTCTGCACAACAAAAACAAGCACACGCATTGTTGGAGGAACAAACTCTTCTTGGGGAGAGTGGCCCTGGCAGGTGAGCCTGCAGGTGAAGCTGACAGCTCAGAGGCACCTGTGTGGAGGGTCACTCATAGGACACCAGTGGGTCCTCACTGCTGCCCACTGCTTTGATGGGCTTCCCCTGCAGGATGTTTGGCGCATCTATAGTGGCATTTTAAATCTGTCAGACATTACAAAAGATACACCTTTCTCACAAATAAAAGAGATTATTATTCACCAAAACTATAAAGTCTCAGAAGGGAATCATGATATCGCCTTGATAAAACTCCAGGCTCCTTTGAATTACACTGAATTCCAAAAACCAATATGCCTACCTTCCAAAGGTGACACAAGCACAATTTATACCAACTGTTGGGTAACCGGATGGGGCTTCTCGAAGGAGAAAGGTGAAATCCAAAATATTCTACAAAAGGTAAATATTCCTTTGGTAACAAATGAAGAATGCCAGAAAAGATATCAAGATTATAAAATAACCCAACGGATGGTCTGTGCTGGCTATAAAGAAGGGGGAAAAGATGCTTGTAAGGGAGATTCAGGTGGTCCCTTAGTTTGCAAACACAATGGAATGTGGCGTTTGGTGGGCATCACCAGCTGGGGTGAAGGCTGTGCCCGCAGGGAGCAACCTGGTGTCTACACCAAAGTCGCTGAGTACATGGACTGGATTTTAGAGAAAACACAGAGCAGTGATGGAAAAGCTCAGATGCAGTCACCAGCATGAGAAGCAGTCCAGAGTCTAGGCAATTTTTACAACCTGAGTTCAAGTCAAATTCTGAGCCTGGGGGGTCCTCATCTGCAAAGCATGGAGAGTGGCATCTTCTTTGCATCCTAAGGACGAAAAACACAGTGCACTCAGAGCTGCTGAGGACAATGTCTGGCTGAAGCCCGCTTTCAGCACGCCGTAACCAGGGGCTGACAATGCGAGGTCGCAACTGAGATCTCCATGACTGTGTGTTGTGAAATAAAATGGTGAAAGATCAAAAAAMouse plasma kallikrein (ACCESSION: NP_032481.1) >gi|6680584|ref|NP_032481.1|kallikrein B, plasma 1 [Mus musculus] (SEQ ID NO: 4)MILFNRVGYFVSLFATVSCGCMTQLYKNTFFRGGDLAAIYTPDAQYCQKMCTFHPRCLLFSFLAVTPPKETNKRFGCFMKESITGTLPRIHRTGAISGHSLKQCGHQISACHRDIYKGLDMRGSNFNISKTDNIEECQKLCTNNFHCQFFTYATSAFYRPEYRKKCLLKHSASGTPTSIKSADNLVSGFSLKSCALSEIGCPMDIFQHSAFADLNVSQVITPDAFVCRTICTFHPNCLFFTFYTNEWETESQRNVCFLKTSKSGRPSPPIPQENAISGYSLLTCRKTRPEPCHSKIYSGVDFEGEELNVTFVQGADVCQETCTKTIRCQFFIYSLLPQDCKEEGCKCSLRLSTDGSPTRITYGMQGSSGYSLRLCKLVDSPDCTTKINARIVGGTNASLGEWPWQVSLQVKLVSQTHLCGGSIIGRQWVLTAAHCFDGIPYPDVWRIYGGILSLSEITKETPSSRIKELIIHQEYKVSEGNYDIALIKLQTPLNYTEFQKPICLPSKADTNTIYTNCWVTGWGYTKEQGETQNILQKATIPLVPNEECQKKYRDYVINKQMICAGYKEGGTDACKGDSGGPLVCKHSGRWQLVGITSWGEGCGRKDQPGVYTKVSEYMDWILEKTQSSDVRALETSSAMouse plasma kallikrein mRNA (ACCESSION: NM_008455.2) >gi|236465804|ref|NM_008455.2|Mus musculus kallikrein B, plasma 1 (Klkb1), mRNA (SEQ ID NO: 5)AGACCGCCCTCGGTGCCATATTCAGAGGGCTTGAAGACCATCTTCATGTGAAGACTCCCTCTCCTCCAGAACCACAACGTGACCATCCTTCCAGGATGATTTTATTCAACCGAGTGGGTTATTTTGTTTCCTTGTTTGCTACCGTCTCCTGTGGGTGTATGACTCAACTGTATAAAAATACCTTCTTCAGAGGTGGGGATCTAGCTGCCATCTACACCCCAGATGCCCAGTACTGTCAGAAGATGTGCACTTTTCACCCCAGGTGCCTGCTGTTCAGCTTTCTCGCCGTGACTCCACCCAAAGAGACAAATAAACGGTTTGGTTGCTTCATGAAAGAGAGCATTACAGGGACTTTGCCAAGAATACACCGGACAGGGGCCATTTCTGGTCATTCTTTAAAGCAGTGTGGCCATCAAATAAGTGCTTGCCACCGAGACATATACAAAGGACTTGATATGAGAGGGTCCAACTTTAATATCTCTAAGACCGACAATATTGAAGAATGCCAGAAACTGTGCACAAATAATTTTCACTGCCAATTTTTCACATATGCTACAAGTGCATTTTACAGACCAGAGTACCGGAAGAAGTGCCTGCTGAAGCACAGTGCAAGCGGAACACCCACCAGCATAAAGTCAGCGGACAACCTGGTGTCTGGATTCTCACTGAAGTCCTGTGCGCTTTCGGAGATAGGTTGCCCCATGGATATTTTCCAGCACTCTGCCTTTGCAGACCTGAATGTAAGCCAGGTCATCACCCCCGATGCCTTTGTGTGTCGCACCATCTGCACCTTCCATCCCAACTGCCTTTTCTTCACGTTCTACACGAATGAATGGGAGACAGAATCACAGAGAAATGTTTGTTTTCTTAAGACGTCTAAAAGTGGAAGACCAAGTCCCCCTATTCCTCAAGAAAACGCTATATCTGGATATAGTCTCCTCACCTGCAGAAAAACTCGCCCTGAACCCTGCCATTCCAAAATTTACTCTGGAGTTGACTTTGAAGGGGAAGAACTGAATGTGACCTTCGTGCAAGGAGCAGATGTCTGCCAAGAGACTTGTACAAAGACAATCCGCTGCCAGTTTTTTATTTACTCCTTACTCCCCCAAGACTGCAAGGAGGAGGGGTGTAAATGTTCCTTAAGGTTATCCACAGATGGCTCCCCAACTAGGATCACCTATGGCATGCAGGGGAGCTCCGGTTATTCTCTGAGATTGTGTAAACTTGTGGACAGCCCTGACTGTACAACAAAAATAAATGCACGTATTGTGGGAGGAACAAACGCTTCTTTAGGGGAGTGGCCATGGCAGGTCAGCCTGCAAGTGAAGCTGGTATCTCAGACCCATTTGTGTGGAGGGTCCATCATTGGTCGCCAATGGGTACTGACAGCTGCCCATTGCTTTGATGGAATTCCCTATCCAGATGTGTGGCGTATATATGGCGGAATTCTTAGTCTGTCCGAGATTACGAAAGAAACGCCTTCCTCGAGAATAAAGGAGCTTATTATTCATCAGGAATACAAAGTCTCAGAAGGCAATTATGATATTGCCTTAATAAAGCTTCAGACGCCCCTGAATTATACTGAATTCCAAAAACCAATATGCCTGCCTTCCAAAGCTGACACAAATACAATTTATACCAACTGTTGGGTGACTGGATGGGGCTACACGAAGGAACAAGGTGAAACGCAAAATATTCTACAAAAGGCTACTATTCCTTTGGTACCAAATGAAGAATGCCAGAAAAAATACAGAGATTATGTTATAAACAAGCAGATGATCTGTGCTGGCTACAAAGAAGGCGGAACAGACGCTTGTAAGGGAGATTCCGGTGGCCCCTTAGTCTGTAAACACAGTGGACGGTGGCAGTTGGTGGGTATCACCAGCTGGGGTGAAGGCTGCGCCCGCAAGGACCAACCAGGAGTCTACACCAAAGTTTCTGAGTACATGGACTGGATATTGGAGAAGACACAGAGCAGTGATGTAAGAGCTCTGGAGACATCTTCAGCCTGAGGAGGCTGGGTACCAAGGAGGAAGAACCCAGCTGGCTTTACCACCTGCCCTCAAGGCAAACTAGAGCTCCAGGATTCTCGGCTGTAAAATGTTGATAATGGTGTCTACCTCACATCCGTATCATTGGATTGAAAATTCAAGTGTAGATATAGTTGCTGAAGACAGCGTTTTGCTCAAGTGTGTTTCCTGCCTTGAGTCACAGGAGCTCCAATGGGAGCATTACAAAGATCACCAAGCTTGTTAGGAAAGAGAATGATCAAAGGGTTTTATTAGGTAATGAAATGTCTAGATGTGATGCAATTGAAAAAAAGACCCCAGATTCTAGCACAGTCCTTGGGACCATTCTCATGTAACTGTTGACTCTGGACCTCAGCAGATCTCAGAGTTACCTGTCCACTTCTGACATTTGTTTATTAGAGCCTGATGCTATTCTTTCAAGTGGAGCAAAAAAAAAAAAAAARat plasma kallikrein (ACCESSION: NP_036857.2) >gi|162138905|ref|NP_036857.2|kallikrein B, plasma 1 [Rattus norvegious] (SEQ ID NO: 6)MILFKQVGYFVSLFATVSCGCLSQLYANTFFRGGDLAAIYTPDAQHCQKMCTFHPRCLLFSFLAVSPTKETDKRFGCFMKESITGTLPRIHRTGAISGHSLKQCGHQLSACHQDIYEGLDMRGSNFNISKTDSIEECQKLCTNNIHCQFFTYATKAFHRPEYRKSCLLKRSSSGTPTSIKPVDNLVSGFSLKSCALSEIGCPMDIFQHFAFADLNVSHVVTPDAFVCRTVCTFHPNCLFFTFYTNEWETESQRNVCFLKTSKSGRPSPPIIQENAVSGYSLFTCRKARPEPCHFKIYSGVAFEGEELNATFVQGADACQETCTKTIRCQFFTYSLLPQDCKAEGCKCSLRLSTDGSPTRITYEAQGSSGYSLRLCKVVESSDCTTKINARIVGGTNSSLGEWPWQVSLQVKLVSQNHMCGGSIIGRQWILTAAHCFDGIPYPDVWRIYGGILNLSEITNKTPFSSIKELIIHQKYKMSEGSYDIALIKLQTPLNYTEFQKPICLPSKADTNTIYTNCWVTGWGYTKERGETQNILQKATIPLVPNEECQKKYRDYVITKQMICAGYKEGGIDACKGDSGGPLVCKHSGRWQLVGITSWGEGCARKEQPGVYTKVAEYIDWILEKIQSSKERALETSPARat plasma kallikrein mRNA (ACCESSION: NM_012725) >gi|162138904|ref|NM_012725.2|Rattus norvegicus kallikrein B, plasma 1 (Klkb1), mRNA (SEQ ID NO: 7)TGAAGACTAGCTTCATGTGAAGACTCCTTCTCCTCCAGCAGCACAAAGCAACCATCCTTCCAGGATGATTTTATTCAAACAAGTGGGTTATTTTGTTTCCTTGTTCGCTACAGTTTCCTGTGGGTGTCTGTCACAACTGTATGCAAATACCTTCTTCAGAGGTGGGGATCTGGCTGCCATCTACACCCCGGATGCCCAGCACTGTCAGAAGATGTGCACGTTTCACCCCAGGTGCCTGCTCTTCAGCTTCCTTGCCGTGAGTCCAACCAAGGAGACAGATAAAAGGTTTGGGTGCTTCATGAAAGAGAGCATTACAGGGACTTTGCCAAGAATACACCGGACAGGGGCCATTTCTGGTCATTCTTTAAAACAGTGTGGCCATCAATTAAGTGCTTGCCACCAAGACATATACGAAGGACTGGATATGAGAGGGTCCAACTTTAATATATCTAAGACCGACAGTATTGAAGAATGCCAGAAACTGTGCACAAATAATATTCACTGCCAATTTTTCACATATGCTACAAAAGCATTTCACAGACCAGAGTACAGGAAGAGTTGCCTGCTGAAGCGCAGTTCAAGTGGAACGCCCACCAGTATAAAGCCAGTGGACAACCTGGTGTCTGGATTCTCACTGAAGTCCTGTGCTCTCTCAGAGATCGGTTGCCCCATGGATATTTTCCAGCACTTTGCCTTTGCAGACCTGAATGTAAGCCATGTCGTCACCCCCGATGCCTTCGTGTGTCGCACCGTTTGCACCTTCCATCCCAACTGCCTCTTCTTCACATTCTACACGAATGAGTGGGAGACGGAATCACAGAGGAATGTTTGTTTTCTTAAGACATCTAAAAGTGGAAGACCAAGTCCCCCTATTATTCAAGAAAATGCTGTATCTGGATACAGTCTCTTCACCTGCAGAAAAGCTCGCCCTGAACCCTGCCATTTCAAGATTTACTCTGGAGTTGCCTTCGAAGGGGAAGAACTGAACGCGACCTTCGTGCAGGGAGCAGATGCGTGCCAAGAGACTTGTACAAAGACCATCCGCTGTCAGTTTTTTACTTACTCATTGCTTCCCCAAGACTGCAAGGCAGAGGGGTGTAAATGTTCCTTAAGGTTATCCACGGATGGCTCTCCAACTAGGATCACCTATGAGGCACAGGGGAGCTCTGGTTATTCTCTGAGACTGTGTAAAGTTGTGGAGAGCTCTGACTGTACGACAAAAATAAATGCACGTATTGTGGGAGGAACAAACTCTTCTTTAGGAGAGTGGCCATGGCAGGTCAGCCTGCAAGTAAAGTTGGTTTCTCAGAATCATATGTGTGGAGGGTCCATCATTGGACGCCAATGGATACTGACGGCTGCCCATTGCTTTGATGGGATTCCCTATCCAGACGTGTGGCGTATATATGGCGGGATTCTTAATCTGTCAGAGATTACAAACAAAACGCCTTTCTCAAGTATAAAGGAGCTTATTATTCATCAGAAATACAAAATGTCAGAAGGCAGTTACGATATTGCCTTAATAAAGCTTCAGACACCGTTGAATTATACTGAATTCCAAAAACCAATATGCCTGCCTTCCAAAGCTGACACAAATACAATTTATACCAACTGCTGGGTGACTGGATGGGGCTACACAAAGGAACGAGGTGAGACCCAAAATATTCTACAAAAGGCAACTATTCCCTTGGTACCAAATGAAGAATGCCAGAAAAAATATAGAGATTATGTTATAACCAAGCAGATGATCTGTGCTGGCTACAAAGAAGGTGGAATAGATGCTTGTAAGGGAGATTCCGGTGGCCCCTTAGTTTGCAAACATAGTGGAAGGTGGCAGTTGGTGGGTATCACCAGCTGGGGCGAAGGCTGTGCCCGCAAGGAGCAACCAGGAGTCTACACCAAAGTTGCTGAGTACATTGACTGGATATTGGAGAAGATACAGAGCAGCAAGGAAAGAGCTCTGGAGACATCTCCAGCATGAGGAGGCTGGGTACTGATGGGGAAGAGCCCAGCTGGCACCAGCTTTACCACCTGCCCTCAAGTCCTACTAGAGCTCCAGAGTTCTCTTCTGCAAAATGTCGATAGTGGTGTCTACCTCGCATCCTTACCATAGGATTAAAAGTCCAAATGTAGACACAGTTGCTAAAGACAGCGCCATGCTCAAGCGTGCTTCCTGCCTTGAGCAACAGGAACGCCAATGAGAACTATCCAAAGATTACCAAGCCTGTTTGGAAATAAAATGGTCAAAGGATTTTTATTAGGTAGTGAAATTAGGTAGTTGTCCTTGGAACCATTCTCATGTAACTGTTGACTCTGGACCTCAGCAGATCACAGTTACCTTCTGTCCACTTCTGACATTTGTGTACTGGAACCTGATGCTGTTCTTCCACTTGGAGCAAAGAACTGAGAAACCTGGTTCTATCCATTGGGAAAAAGAGATCTTTGTAACATTTCCTTTACAATAAAAAGATGTTCTACTTGGACTTGAAAAAAAAAAAAAAAAAAAAAAAAAA

Display Libraries

A display library is a collection of entities; each entity includes anaccessible polypeptide component and a recoverable component thatencodes or identifies the polypeptide component. The polypeptidecomponent is varied so that different amino acid sequences arerepresented. The polypeptide component can be of any length, e.g. fromthree amino acids to over 300 amino acids. A display library entity caninclude more than one polypeptide component, for example, the twopolypeptide chains of a sFab. In one exemplary implementation, a displaylibrary can be used to identify proteins that bind to plasma kallikrein.In a selection, the polypeptide component of each member of the libraryis probed with plasma kallikrein (or fragment thereof) and if thepolypeptide component binds to the plasma kallikrein, the displaylibrary member is identified, typically by retention on a support.

Retained display library members are recovered from the support andanalyzed. The analysis can include amplification and a subsequentselection under similar or dissimilar conditions. For example, positiveand negative selections can be alternated. The analysis can also includedetermining the amino acid sequence of the polypeptide component andpurification of the polypeptide component for detailed characterization.

A variety of formats can be used for display libraries. Examples includethe following.

Phage Display:

The protein component is typically covalently linked to a bacteriophagecoat protein. The linkage results from translation of a nucleic acidencoding the protein component fused to the coat protein. The linkagecan include a flexible peptide linker, a protease site, or an amino acidincorporated as a result of suppression of a stop codon. Phage displayis described, for example, in U.S. Pat. No. 5,223,409; Smith (1985)Science 228:1315-1317; WO 92/18619; WO 91/17271; WO 92/20791; WO92/15679; WO 93/01288; WO 92/01047; WO 92/09690; WO 90/02809; de Haardet al. (1999) J. Biol. Chem 274:18218-30; Hoogenboom et al. (1998)Immunotechnology 4:1-20; Hoogenboom et al. (2000) Immunol Today 2:371-8and Hoet et al. (2005) Nat Biotechnol. 23(3)344-8. Bacteriophagedisplaying the protein component can be grown and harvested usingstandard phage preparatory methods, e.g. PEG precipitation from growthmedia. After selection of individual display phages, the nucleic acidencoding the selected protein components can be isolated from cellsinfected with the selected phages or from the phage themselves, afteramplification. Individual colonies or plaques can be picked, the nucleicacid isolated and sequenced.

Other Display Formats.

Other display formats include cell based display (see, e.g., WO03/029456), protein-nucleic acid fusions (see, e.g., U.S. Pat. No.6,207,446), ribosome display (See, e.g., Mattheakis et al. (1994) Proc.Natl. Acad. Sci. USA 91:9022 and Hanes et al. (2000) Nat Biotechnol.18:1287-92; Hanes et al. (2000) Methods Enzymol. 328:404-30; andSchaffitzel et al. (1999) J Immunol Methods. 231(1-2):119-35), and E.coli periplasmic display (J Immunol Methods. 2005 Nov. 22; PMID:16337958).

Scaffolds.

Scaffolds useful for display include: antibodies (e.g., Fab fragments,single chain Fv molecules (scFv), single domain antibodies, camelidantibodies, and camelized antibodies); T-cell receptors; MHC proteins;extracellular domains (e.g., fibronectin Type III repeats, EGF repeats);protease inhibitors (e.g., Kunitz domains, ecotin, BPTI, and so forth);TPR repeats; trifoil structures; zinc finger domains; DNA-bindingproteins; particularly monomeric DNA binding proteins; RNA bindingproteins; enzymes, e.g., proteases (particularly inactivated proteases),RNase; chaperones, e.g., thioredoxin and heat shock proteins;intracellular signaling domains (such as SH2 and SH3 domains); linearand constrained peptides; and linear peptide substrates. Displaylibraries can include synthetic and/or natural diversity. See, e.g.,U.S. 2004-0005709.

Display technology can also be used to obtain binding proteins (e.g.,antibodies) that bind particular epitopes of a target. This can be done,for example, by using competing non-target molecules that lack theparticular epitope or are mutated within the epitope, e.g., withalanine. Such non-target molecules can be used in a negative selectionprocedure as described below, as competing molecules when binding adisplay library to the target, or as a pre-elution agent, e.g., tocapture in a wash solution dissociating display library members that arenot specific to the target.

Iterative Selection.

In one preferred embodiment, display library technology is used in aniterative mode. A first display library is used to identify one or morebinding proteins for a target. These identified binding proteins arethen varied using a mutagenesis method to form a second display library.Higher affinity binding proteins are then selected from the secondlibrary, e.g., by using higher stringency or more competitive bindingand washing conditions.

In some implementations, the mutagenesis is targeted to regions at thebinding interface. If, for example, the identified binding proteins areantibodies, then mutagenesis can be directed to the CDR regions of theheavy or light chains as described herein. Further, mutagenesis can bedirected to framework regions near or adjacent to the CDRs. In the caseof antibodies, mutagenesis can also be limited to one or a few of theCDRs, e.g., to make precise step-wise improvements. Exemplarymutagenesis techniques include: error-prone PCR, recombination, DNAshuffling, site-directed mutagenesis and cassette mutagenesis.

In one example of iterative selection, the methods described herein areused to first identify a protein from a display library that bindsplasma kallikrein, with at least a minimal binding specificity for atarget or a minimal activity, e.g., an equilibrium dissociation constantfor binding of less than 0.5 nM, 1 nM, 10 nM, or 100 nM. The nucleicacid sequences encoding the initial identified proteins are used as atemplate nucleic acid for the introduction of variations, e.g., toidentify a second protein that has enhanced properties (e.g., bindingaffinity, kinetics, or stability) relative to the initial protein.

Off-Rate Selection.

Since a slow dissociation rate can be predictive of high affinity,particularly with respect to interactions between polypeptides and theirtargets, the methods described herein can be used to isolate bindingproteins with a desired (e.g., reduced) kinetic dissociation rate for abinding interaction to a target.

To select for slow dissociating binding proteins from a display library,the library is contacted to an immobilized target. The immobilizedtarget is then washed with a first solution that removesnon-specifically or weakly bound biomolecules. Then the bound bindingproteins are eluted with a second solution that includes a saturatingamount of free target or a target specific high-affinity competingmonoclonal antibody, i.e., replicates of the target that are notattached to the particle. The free target binds to biomolecules thatdissociate from the target. Rebinding is effectively prevented by thesaturating amount of free target relative to the much lowerconcentration of immobilized target.

The second solution can have solution conditions that are substantiallyphysiological or that are stringent. Typically, the solution conditionsof the second solution are identical to the solution conditions of thefirst solution. Fractions of the second solution are collected intemporal order to distinguish early from late fractions. Later fractionsinclude biomolecules that dissociate at a slower rate from the targetthan biomolecules in the early fractions.

Further, it is also possible to recover display library members thatremain bound to the target even after extended incubation. These caneither be dissociated using chaotropic conditions or can be amplifiedwhile attached to the target. For example, phage bound to the target canbe contacted to bacterial cells.

Selecting or Screening for Specificity.

The display library screening methods described herein can include aselection or screening process that discards display library membersthat bind to a non-target molecule. Examples of non-target moleculesinclude streptavidin on magnetic beads, blocking agents such as bovineserum albumin, non-fat bovine milk, soy protein, any capturing or targetimmobilizing monoclonal antibody, or non-transfected cells which do notexpress the target.

In one implementation, a so-called “negative selection” step is used todiscriminate between the target and related non-target molecule and arelated, but distinct non-target molecule. The display library or a poolthereof is contacted to the non-target molecule. Members of the samplethat do not bind the non-target are collected and used in subsequentselections for binding to the target molecule or even for subsequentnegative selections. The negative selection step can be prior to orafter selecting library members that bind to the target molecule.

In another implementation, a screening step is used. After displaylibrary members are isolated for binding to the target molecule, eachisolated library member is tested for its ability to bind to anon-target molecule (e.g., a non-target listed above). For example, ahigh-throughput ELISA screen can be used to obtain this data. The ELISAscreen can also be used to obtain quantitative data for binding of eachlibrary member to the target as well as for cross species reactivity torelated targets or subunits of the target (e.g., plasma kallikrein) andalso under different condition such as pH 6 or pH 7.5. The non-targetand target binding data are compared (e.g., using a computer andsoftware) to identify library members that specifically bind to thetarget.

Other Exemplary Expression Libraries

Other types of collections of proteins (e.g., expression libraries) canbe used to identify proteins with a particular property (e.g., abilityto bind plasma kallikrein), including, e.g., protein arrays ofantibodies (see, e.g., De Wildt et al. (2000) Nat. Biotechnol.18:989-994), lambda gtll libraries, two-hybrid libraries and so forth.

Exemplary Libraries

It is possible to immunize a non-human primate and recover primateantibody genes that can be displayed on phage (see below). From such alibrary, one can select antibodies that bind the antigen used inimmunization. See, for example, Vaccine. (2003) 22(2):257-67 orImmunogenetics. (2005) 57(10):730-8. Thus one could obtain primateantibodies that bind and inhibit plasma kallikrein by immunizing achimpanzee or macaque and using a variety of means to select or screenfor primate antibodies that bind and inhibit plasma kallikrein. One canalso make chimeras of primatized Fabs with human constant regions, seeCurr Opin Mol Ther. (2004) 6(6):675-83. “PRIMATIZED antibodies,genetically engineered from cynomolgus macaque monkey and humancomponents, are structurally indistinguishable from human antibodies.They may, therefore, be less likely to cause adverse reactions inhumans, making them potentially suited for long-term, chronic treatment”Curr Opin Investig Drugs. (2001) 2(5):635-8.

One exemplary type of library presents a diverse pool of polypeptides,each of which includes an immunoglobulin domain, e.g., an immunoglobulinvariable domain. Of interest are display libraries where the members ofthe library include primate or “primatized” (e.g., such as human,non-human primate or “humanized”) immunoglobin domains (e.g.,immunoglobin variable domains) or chimeric primatized Fabs with humanconstant regions. Human or humanized immunoglobin domain libraries maybe used to identify human or “humanized” antibodies that, for example,recognize human antigens. Because the constant and framework regions ofthe antibody are human, these antibodies may avoid themselves beingrecognized and targeted as antigens when administered to humans. Theconstant regions may also be optimized to recruit effector functions ofthe human immune system. The in vitro display selection processsurmounts the inability of a normal human immune system to generateantibodies against self-antigens.

A typical antibody display library displays a polypeptide that includesa VH domain and a VL domain. An “immunoglobulin domain” refers to adomain from the variable or constant domain of immunoglobulin molecules.Immunoglobulin domains typically contain two β-sheets formed of aboutseven β-strands, and a conserved disulphide bond (see, e.g., A. F.Williams and A. N. Barclay, 1988, Ann. Rev. Immunol. 6:381-405). Thedisplay library can display the antibody as a Fab fragment (e.g., usingtwo polypeptide chains) or a single chain Fv (e.g., using a singlepolypeptide chain). Other formats can also be used.

As in the case of the Fab and other formats, the displayed antibody caninclude one or more constant regions as part of a light and/or heavychain. In one embodiment, each chain includes one constant region, e.g.,as in the case of a Fab. In other embodiments, additional constantregions are displayed.

Antibody libraries can be constructed by a number of processes (see,e.g., de Haard et al., 1999, J. Biol. Chem. 274:18218-30; Hoogenboom etal., 1998, Immunotechnology 4:1-20; Hoogenboom et al., 2000, Immunol.Today 21:371-378, and Hoet et al. (2005) Nat Biotechnol. 23(3):344-8.Further, elements of each process can be combined with those of otherprocesses. The processes can be used such that variation is introducedinto a single immunoglobulin domain (e.g., VH or VL) or into multipleimmunoglobulin domains (e.g., VH and VL). The variation can beintroduced into an immunoglobulin variable domain, e.g., in the regionof one or more of CDR1, CDR2, CDR3, FR1, FR2, FR3, and/or FR4, referringto such regions of either and both of heavy and light chain variabledomains. For example, the variation(s) may be introduced into all threeCDRs of a given variable domain, or into CDR1 and CDR2, e.g., of a heavychain variable domain. Any combination is feasible. In one process,antibody libraries are constructed by inserting diverse oligonucleotidesthat encode CDRs into the corresponding regions of the nucleic acid. Theoligonucleotides can be synthesized using monomeric nucleotides ortrinucleotides. For example, Knappik et al., 2000, J. Mol. Biol.296:57-86 describe a method for constructing CDR encodingoligonucleotides using trinucleotide synthesis and a template withengineered restriction sites for accepting the oligonucleotides.

In another process, an animal (e.g., a rodent) is immunized with plasmakallikrein. The animal is optionally boosted with the antigen to furtherstimulate the response. Then spleen cells are isolated from the animal,and nucleic acid encoding VH and/or VL domains is amplified and clonedfor expression in the display library.

In yet another process, antibody libraries are constructed from nucleicacid amplified from naïve germline immunoglobulin genes. The amplifiednucleic acid includes nucleic acid encoding the VH and/or VL domain.Sources of immunoglobulin-encoding nucleic acids are described below.Amplification can include PCR, e.g., with primers that anneal to theconserved constant region, or another amplification method.

Nucleic acid encoding immunoglobulin domains can be obtained from theimmune cells of, e.g., a primate (e.g., a human), mouse, rabbit, camel,or rodent. In one example, the cells are selected for a particularproperty. B cells at various stages of maturity can be selected. Inanother example, the B cells are naïve.

In one embodiment, fluorescent-activated cell sorting (FACS) is used tosort B cells that express surface-bound IgM, IgD, or IgG molecules.Further, B cells expressing different isotypes of IgG can be isolated.In another preferred embodiment, the B or T cells are cultured in vitro.The cells can be stimulated in vitro, e.g., by culturing with feedercells or by adding mitogens or other modulatory reagents, such asantibodies to CD40, CD40 ligand or CD20, phorbol myristate acetate,bacterial lipopolysaccharide, concanavalin A, phytohemagglutinin, orpokeweed mitogen.

In another embodiment, the cells are isolated from a subject that has adisease of condition described herein, e.g., a plasma kallikreinassociated disease or condition.

In one preferred embodiment, the cells have activated a program ofsomatic hypermutation. Cells can be stimulated to undergo somaticmutagenesis of immunoglobulin genes, for example, by treatment withanti-immunoglobulin, anti-CD40, and anti-CD38 antibodies (see, e.g.,Bergthorsdottir et al., 2001, J. Immunol. 166:2228). In anotherembodiment, the cells are naïve.

The nucleic acid encoding an immunoglobulin variable domain can beisolated from a natural repertoire by the following exemplary method.First, RNA is isolated from the immune cell. Full length (i.e., capped)mRNAs are separated (e.g. by degrading uncapped RNAs with calfintestinal phosphatase). The cap is then removed with tobacco acidpyrophosphatase and reverse transcription is used to produce the cDNAs.

The reverse transcription of the first (antisense) strand can be done inany manner with any suitable primer. See, e.g., de Haard et al., 1999,J. Biol. Chem. 274:18218-30. The primer binding region can be constantamong different immunoglobulins, e.g., in order to reverse transcribedifferent isotypes of immunoglobulin. The primer binding region can alsobe specific to a particular isotype of immunoglobulin. Typically, theprimer is specific for a region that is 3′ to a sequence encoding atleast one CDR. In another embodiment, poly-dT primers may be used (andmay be preferred for the heavy-chain genes).

A synthetic sequence can be ligated to the 3′ end of the reversetranscribed strand. The synthetic sequence can be used as a primerbinding site for binding of the forward primer during PCR amplificationafter reverse transcription. The use of the synthetic sequence canobviate the need to use a pool of different forward primers to fullycapture the available diversity.

The variable domain-encoding gene is then amplified, e.g., using one ormore rounds. If multiple rounds are used, nested primers can be used forincreased fidelity. The amplified nucleic acid is then cloned into adisplay library vector.

Secondary Screening Methods

After selecting candidate library members that bind to a target, eachcandidate library member can be further analyzed, e.g., to furthercharacterize its binding properties for the target, e.g., plasmakallikrein. Each candidate library member can be subjected to one ormore secondary screening assays. The assay can be for a bindingproperty, a catalytic property, an inhibitory property, a physiologicalproperty (e.g., cytotoxicity, renal clearance, immunogenicity), astructural property (e.g., stability, conformation, oligomerizationstate) or another functional property. The same assay can be usedrepeatedly, but with varying conditions, e.g., to determine pH, ionic,or thermal sensitivities.

As appropriate, the assays can use a display library member directly, arecombinant polypeptide produced from the nucleic acid encoding theselected polypeptide, or a synthetic peptide synthesized based on thesequence of the selected polypeptide. In the case of selected Fabs, theFabs can be evaluated or can be modified and produced as intact IgGproteins. Exemplary assays for binding properties include the following.

ELISA.

Binding proteins can be evaluated using an ELISA assay. For example,each protein is contacted to a microtitre plate whose bottom surface hasbeen coated with the target, e.g., a limiting amount of the target. Theplate is washed with buffer to remove non-specifically boundpolypeptides. Then the amount of the binding protein bound to the targeton the plate is determined by probing the plate with an antibody thatcan recognize the binding protein, e.g., a tag or constant portion ofthe binding protein. The antibody is linked to a detection system (e.g.,an enzyme such as alkaline phosphatase or horse radish peroxidase (HRP)which produces a colorimetric product when appropriate substrates areprovided).

Homogeneous Binding Assays.

The ability of a binding protein described herein to bind a target canbe analyzed using a homogenous assay, i.e., after all components of theassay are added, additional fluid manipulations are not required. Forexample, fluorescence resonance energy transfer (FRET) can be used as ahomogenous assay (see, for example, Lakowicz et al., U.S. Pat. No.5,631,169; Stavrianopoulos, et al., U.S. Pat. No. 4,868,103). Afluorophore label on the first molecule (e.g., the molecule identifiedin the fraction) is selected such that its emitted fluorescent energycan be absorbed by a fluorescent label on a second molecule (e.g., thetarget) if the second molecule is in proximity to the first molecule.The fluorescent label on the second molecule fluoresces when it absorbsto the transferred energy. Since the efficiency of energy transferbetween the labels is related to the distance separating the molecules,the spatial relationship between the molecules can be assessed. In asituation in which binding occurs between the molecules, the fluorescentemission of the ‘acceptor’ molecule label in the assay should bemaximal. A binding event that is configured for monitoring by FRET canbe conveniently measured through standard fluorometric detection means,e.g., using a fluorimeter. By titrating the amount of the first orsecond binding molecule, a binding curve can be generated to estimatethe equilibrium binding constant.

Another example of a homogenous assay is ALPHASCREEN™ (PackardBioscience, Meriden Conn.). ALPHASCREEN™ uses two labeled beads. Onebead generates singlet oxygen when excited by a laser. The other beadgenerates a light signal when singlet oxygen diffuses from the firstbead and collides with it. The signal is only generated when the twobeads are in proximity. One bead can be attached to the display librarymember, the other to the target. Signals are measured to determine theextent of binding.

Surface Plasmon Resonance (SPR).

The interaction of binding protein and a target can be analyzed usingSPR. SPR or Biomolecular Interaction Analysis (BIA) detects biospecificinteractions in real time, without labeling any of the interactants.Changes in the mass at the binding surface (indicative of a bindingevent) of the BIA chip result in alterations of the refractive index oflight near the surface (the optical phenomenon of surface plasmonresonance (SPR)). The changes in the refractivity generate a detectablesignal, which are measured as an indication of real-time reactionsbetween biological molecules. Methods for using SPR are described, forexample, in U.S. Pat. No. 5,641,640; Raether, 1988, Surface PlasmonsSpringer Verlag; Sjolander and Urbaniczky, 1991, Anal. Chem.63:2338-2345; Szabo et al., 1995, Curr. Opin. Struct. Biol. 5:699-705and on-line resources provide by BIAcore International AB (Uppsala,Sweden).

Information from SPR can be used to provide an accurate and quantitativemeasure of the equilibrium dissociation constant (K_(D)), and kineticparameters, including K_(on) and K_(off), for the binding of a bindingprotein to a target. Such data can be used to compare differentbiomolecules. For example, selected proteins from an expression librarycan be compared to identify proteins that have high affinity for thetarget or that have a slow K_(off). This information can also be used todevelop structure-activity relationships (SAR). For example, the kineticand equilibrium binding parameters of matured versions of a parentprotein can be compared to the parameters of the parent protein. Variantamino acids at given positions can be identified that correlate withparticular binding parameters, e.g., high affinity and slow K_(off).This information can be combined with structural modeling (e.g., usinghomology modeling, energy minimization, or structure determination byx-ray crystallography or NMR). As a result, an understanding of thephysical interaction between the protein and its target can beformulated and used to guide other design processes.

Cellular Assays.

Binding proteins can be screened for ability to bind to cells whichtransiently or stably express and display the target of interest on thecell surface. For example, plasma kallikrein binding proteins can befluorescently labeled and binding to plasma kallikrein in the presenceof absence of antagonistic antibody can be detected by a change influorescence intensity using flow cytometry e.g., a FACS machine.

Other Exemplary Methods for Obtaining Plasma Kallikrein Binding Proteins

In addition to the use of display libraries, other methods can be usedto obtain a plasma kallikrein binding protein (e.g., antibody). Forexample, plasma kallikrein protein or a fragment thereof can be used asan antigen in a non-human animal, e.g., a rodent.

In one embodiment, the non-human animal includes at least a part of ahuman immunoglobulin gene. For example, it is possible to engineer mousestrains deficient in mouse antibody production with large fragments ofthe human Ig loci. Using the hybridoma technology, antigen-specificmonoclonal antibodies (Mabs) derived from the genes with the desiredspecificity may be produced and selected. See, e.g., XENOMOUSE™, Greenet al., 1994, Nat. Gen. 7:13-21; U.S. 2003-0070185, WO 96/34096,published Oct. 31, 1996, and PCT Application No. PCT/US96/05928, filedApr. 29, 1996.

In another embodiment, a monoclonal antibody is obtained from thenon-human animal, and then modified, e.g., humanized or deimmunized.Winter describes a CDR-grafting method that may be used to prepare thehumanized antibodies (UK Patent Application GB 2188638A, filed on Mar.26, 1987; U.S. Pat. No. 5,225,539. All of the CDRs of a particular humanantibody may be replaced with at least a portion of a non-human CDR oronly some of the CDRs may be replaced with non-human CDRs. It is onlynecessary to replace the number of CDRs required for binding of thehumanized antibody to a predetermined antigen.

Humanized antibodies can be generated by replacing sequences of the Fvvariable region that are not directly involved in antigen binding withequivalent sequences from human Fv variable regions. General methods forgenerating humanized antibodies are provided by Morrison, S. L., 1985,Science 229:1202-1207, by Oi et al., 1986, BioTechniques 4:214, and byQueen et al. U.S. Pat. Nos. 5,585,089, 5,693,761 and 5,693,762. Thosemethods include isolating, manipulating, and expressing the nucleic acidsequences that encode all or part of immunoglobulin Fv variable regionsfrom at least one of a heavy or light chain. Numerous sources of suchnucleic acid are available. For example, nucleic acids may be obtainedfrom a hybridoma producing an antibody against a predetermined target,as described above. The recombinant DNA encoding the humanized antibody,or fragment thereof, can then be cloned into an appropriate expressionvector.

Reducing Immunogenicity of Plasma Kallikrein Binding Proteins

Immunoglobin plasma kallikrein binding proteins (e.g., IgG or Fab plasmakallikrein binding proteins) may be modified to reduce immunogenicity.Reduced immunogenicity is desirable in plasma kallikrein bindingproteins intended for use as therapeutics, as it reduces the chance thatthe subject will develop an immune response against the therapeuticmolecule. Techniques useful for reducing immunogenicity of plasmakallikrein binding proteins include deletion/modification of potentialhuman T cell epitopes and “germlining” of sequences outside of the CDRs(e.g., framework and Fc).

A plasma kallikrein-binding antibody may be modified by specificdeletion of human T cell epitopes or “deimmunization,” e.g., by themethods disclosed in WO 98/52976 and WO 00/34317. Briefly, the heavy andlight chain variable regions of an antibody are analyzed for peptidesthat bind to MHC Class II; these peptides represent potential T-cellepitopes (as defined in WO 98/52976 and WO 00/34317). For detection ofpotential T-cell epitopes, a computer modeling approach termed “peptidethreading” can be applied, and in addition a database of human MHC classII binding peptides can be searched for motifs present in the VH and VLsequences, as described in WO 98/52976 and WO 00/34317. These motifsbind to any of the 18 major MHC class II DR allotypes, and thusconstitute potential T cell epitopes. Potential T-cell epitopes detectedcan be eliminated by substituting small numbers of amino acid residuesin the variable regions, or preferably, by single amino acidsubstitutions. As far as possible conservative substitutions are made,often but not exclusively, an amino acid common at this position inhuman germline antibody sequences may be used. Human germline sequencesare disclosed in Tomlinson, I. A. et al., 1992, J. Mol. Biol.227:776-798; Cook, G. P. et al., 1995, Immunol. Today Vol. 16 (5):237-242; Chothia, D. et al., 1992, J. Mol. Bio. 227:799-817. The V BASEdirectory provides a comprehensive directory of human immunoglobulinvariable region sequences (compiled by Tomlinson, I. A. et al. MRCCentre for Protein Engineering, Cambridge, UK). After the deimmunizingchanges are identified, nucleic acids encoding V_(H) and V_(L) can beconstructed by mutagenesis or other synthetic methods (e.g., de novosynthesis, cassette replacement, and so forth). Mutagenized variablesequence can, optionally, be fused to a human constant region, e.g.,human IgG1 or κ constant regions.

In some cases a potential T cell epitope will include residues which areknown or predicted to be important for antibody function. For example,potential T cell epitopes are usually biased towards the CDRs. Inaddition, potential T cell epitopes can occur in framework residuesimportant for antibody structure and binding. Changes to eliminate thesepotential epitopes will in some cases require more scrutiny, e.g., bymaking and testing chains with and without the change. Where possible,potential T cell epitopes that overlap the CDRs were eliminated bysubstitutions outside the CDRs. In some cases, an alteration within aCDR is the only option, and thus variants with and without thissubstitution should be tested. In other cases, the substitution requiredto remove a potential T cell epitope is at a residue position within theframework that might be critical for antibody binding. In these cases,variants with and without this substitution should be tested. Thus, insome cases several variant deimmunized heavy and light chain variableregions were designed and various heavy/light chain combinations testedin order to identify the optimal deimmunized antibody. The choice of thefinal deimmunized antibody can then be made by considering the bindingaffinity of the different variants in conjunction with the extent ofdeimmunization, i.e., the number of potential T cell epitopes remainingin the variable region. Deimmunization can be used to modify anyantibody, e.g., an antibody that includes a non-human sequence, e.g., asynthetic antibody, a murine antibody other non-human monoclonalantibody, or an antibody isolated from a display library.

Plasma kallikrein binding antibodies are “germlined” by reverting one ormore non-germline amino acids in framework regions to correspondinggermline amino acids of the antibody, so long as binding properties aresubstantially retained. Similar methods can also be used in the constantregion, e.g., in constant immunoglobulin domains.

Antibodies that bind to plasma kallikrein, e.g., an antibody describedherein, may be modified in order to make the variable regions of theantibody more similar to one or more germline sequences. For example, anantibody can include one, two, three, or more amino acid substitutions,e.g., in a framework, CDR, or constant region, to make it more similarto a reference germline sequence. One exemplary germlining method caninclude identifying one or more germline sequences that are similar(e.g., most similar in a particular database) to the sequence of theisolated antibody. Mutations (at the amino acid level) are then made inthe isolated antibody, either incrementally or in combination with othermutations. For example, a nucleic acid library that includes sequencesencoding some or all possible germline mutations is made. The mutatedantibodies are then evaluated, e.g., to identify an antibody that hasone or more additional germline residues relative to the isolatedantibody and that is still useful (e.g., has a functional activity). Inone embodiment, as many germline residues are introduced into anisolated antibody as possible.

In one embodiment, mutagenesis is used to substitute or insert one ormore germline residues into a framework and/or constant region. Forexample, a germline framework and/or constant region residue can be froma germline sequence that is similar (e.g., most similar) to thenon-variable region being modified. After mutagenesis, activity (e.g.,binding or other functional activity) of the antibody can be evaluatedto determine if the germline residue or residues are tolerated (i.e., donot abrogate activity). Similar mutagenesis can be performed in theframework regions.

Selecting a germline sequence can be performed in different ways. Forexample, a germline sequence can be selected if it meets a predeterminedcriteria for selectivity or similarity, e.g., at least a certainpercentage identity, e.g., at least 75, 80, 85, 90, 91, 92, 93, 94, 95,96, 97, 98, 99, or 99.5% identity. The selection can be performed usingat least 2, 3, 5, or 10 germline sequences. In the case of CDR1 andCDR2, identifying a similar germline sequence can include selecting onesuch sequence. In the case of CDR3, identifying a similar germlinesequence can include selecting one such sequence, but may include usingtwo germline sequences that separately contribute to the amino-terminalportion and the carboxy-terminal portion of the sequence. In otherimplementations more than one or two germline sequences are used, e.g.,to form a consensus sequence.

In one embodiment, with respect to a particular reference variabledomain sequence, e.g., a sequence described herein, a related variabledomain sequence has at least 30, 40, 50, 60, 70, 80, 90, 95 or 100% ofthe CDR amino acid positions that are not identical to residues in thereference CDR sequences, residues that are identical to residues atcorresponding positions in a human germline sequence (i.e., an aminoacid sequence encoded by a human germline nucleic acid).

In one embodiment, with respect to a particular reference variabledomain sequence, e.g., a sequence described herein, a related variabledomain sequence has at least 30, 50, 60, 70, 80, 90 or 100% of the FRregions identical to FR sequence from a human germline sequence, e.g., agermline sequence related to the reference variable domain sequence.

Accordingly, it is possible to isolate an antibody which has similaractivity to a given antibody of interest, but is more similar to one ormore germline sequences, particularly one or more human germlinesequences. For example, an antibody can be at least 90, 91, 92, 93, 94,95, 96, 97, 98, 99, or 99.5% identical to a germline sequence in aregion outside the CDRs (e.g., framework regions). Further, an antibodycan include at least 1, 2, 3, 4, or 5 germline residues in a CDR region,the germline residue being from a germline sequence of similar (e.g.,most similar) to the variable region being modified. Germline sequencesof primary interest are human germline sequences. The activity of theantibody (e.g., the binding activity as measured by K_(A)) can be withina factor or 100, 10, 5, 2, 0.5, 0.1, and 0.001 of the original antibody.

Germline sequences of human immunoglobin genes have been determined andare available from a number of sources, including the INTERNATIONALIMMUNOGENETICS INFORMATION SYSTEM® (IMGT), and the V BASE directory(compiled by Tomlinson, I. A. et al. MRC Centre for Protein Engineering,Cambridge, UK).

Exemplary germline reference sequences for V_(kappa) include: O12/O2,O18/O8, A20, A30, L14, L1, L15, L4/18a, L5/L19, L8, L23, L9, L24, L11,L12, O11/O1, A17, A1, A18, A2, A19/A3, A23, A27, A11, L2/L16, L6, L20,L25, B3, B2, A26/A10, and A14. See, e.g., Tomlinson et al., 1995, EMBOJ. 14(18):4628-3.

A germline reference sequence for the HC variable domain can be based ona sequence that has particular canonical structures, e.g., 1-3structures in the H1 and H2 hypervariable loops. The canonicalstructures of hypervariable loops of an immunoglobulin variable domaincan be inferred from its sequence, as described in Chothia et al., 1992,J. Mol. Biol. 227:799-817; Tomlinson et al., 1992, J. Mol. Biol.227:776-798); and Tomlinson et al., 1995, EMBO J. 14(18):4628-38.Exemplary sequences with a 1-3 structure include: DP-1, DP-8, DP-12,DP-2, DP-25, DP-15, DP-7, DP-4, DP-31, DP-32, DP-33, DP-35, DP-40, 7-2,hv3005, hv3005f3, DP-46, DP-47, DP-58, DP-49, DP-50, DP-51, DP-53, andDP-54.

Protein Production

Standard recombinant nucleic acid methods can be used to express aprotein that binds to plasma kallikrein. Generally, a nucleic acidsequence encoding the protein is cloned into a nucleic acid expressionvector. Of course, if the protein includes multiple polypeptide chains,each chain can be cloned into an expression vector, e.g., the same ordifferent vectors, that are expressed in the same or different cells.

Antibody Production.

Some antibodies, e.g., Fabs, can be produced in bacterial cells, e.g.,E. coli cells (see e.g., Nadkarni, A. et al., 2007 Protein Expr Purif52(1):219-29). For example, if the Fab is encoded by sequences in aphage display vector that includes a suppressible stop codon between thedisplay entity and a bacteriophage protein (or fragment thereof), thevector nucleic acid can be transferred into a bacterial cell that cannotsuppress a stop codon. In this case, the Fab is not fused to the geneIII protein and is secreted into the periplasm and/or media.

Antibodies can also be produced in eukaryotic cells. In one embodiment,the antibodies (e.g., scFv's) are expressed in a yeast cell such asPichia (see, e.g., Powers et al., 2001, J. Immunol. Methods. 251:123-35;Schoonooghe S. et al., 2009 BMC Biotechnol. 9:70; Abdel-Salam, H A. etal., 2001 Appl Microbiol Biotechnol 56(1-2):157-64; Takahashi K. et al.,2000 Biosci Biotechnol Biochem 64(10):2138-44; Edqvist, J. et al., 1991J Biotechnol 20(3):291-300), Hanseula, or Saccharomyces. One of skill inthe art can optimize antibody production in yeast by optimizing, forexample, oxygen conditions (see e.g., Baumann K., et al. 2010 BMC Syst.Biol. 4:141), osmolarity (see e.g., Dragosits, M. et al., 2010 BMCGenomics 11:207), temperature (see e.g., Dragosits, M. et al., 2009 JProteome Res. 8(3):1380-92), fermentation conditions (see e.g., Ning, D.et al. 2005 J. Biochem. and Mol. Biol. 38(3): 294-299), strain of yeast(see e.g., Kozyr, A V et al. 2004 Mol Biol (Mosk) 38(6):1067-75;Horwitz, A H. et al., 1988 Proc Natl Acad Sci USA 85(22):8678-82;Bowdish, K. et al. 1991 J Biol Chem 266(18):11901-8), overexpression ofproteins to enhance antibody production (see e.g., Gasser, B. et al.,2006 Biotechol. Bioeng. 94(2):353-61), level of acidity of the culture(see e.g., Kobayashi H., et al., 1997 FEMS Microbiol Lett152(2):235-42), concentrations of substrates and/or ions (see e.g., Ko JH. et al., 2996 Appl Biochem Biotechnol 60(1):41-8). In addition, yeastsystems can be used to produce antibodies with an extended half-life(see e.g., Smith, B J. et al. 2001 Bioconjug Chem 12(5):750-756),

In one preferred embodiment, antibodies are produced in mammalian cells.Preferred mammalian host cells for expressing the clone antibodies orantigen-binding fragments thereof include Chinese Hamster Ovary (CHOcells) (including dhfr− CHO cells, described in Urlaub and Chasin, 1980,Proc. Natl. Acad. Sci. USA 77:4216-4220, used with a DHFR selectablemarker, e.g., as described in Kaufman and Sharp, 1982, Mol. Biol.159:601 621), lymphocytic cell lines, e.g., NSO myeloma cells and SP2cells, COS cells, HEK293T cells (J. Immunol. Methods (2004)289(1-2):65-80), and a cell from a transgenic animal, e.g., a transgenicmammal. For example, the cell is a mammary epithelial cell.

In some embodiments, plasma kallikrein binding proteins are produced ina plant or cell-free based system (see e.g., Galeffi, P., et al., 2006 JTransl Med 4:39).

In addition to the nucleic acid sequence encoding the diversifiedimmunoglobulin domain, the recombinant expression vectors may carryadditional sequences, such as sequences that regulate replication of thevector in host cells (e.g., origins of replication) and selectablemarker genes. The selectable marker gene facilitates selection of hostcells into which the vector has been introduced (see e.g., U.S. Pat.Nos. 4,399,216, 4,634,665 and 5,179,017). For example, typically theselectable marker gene confers resistance to drugs, such as G418,hygromycin or methotrexate, on a host cell into which the vector hasbeen introduced. Preferred selectable marker genes include thedihydrofolate reductase (DHFR) gene (for use in dhfr⁻ host cells withmethotrexate selection/amplification) and the neo gene (for G418selection).

In an exemplary system for recombinant expression of an antibody, orantigen-binding portion thereof, a recombinant expression vectorencoding both the antibody heavy chain and the antibody light chain isintroduced into dhfr⁻CHO cells by calcium phosphate-mediatedtransfection. Within the recombinant expression vector, the antibodyheavy and light chain genes are each operatively linked toenhancer/promoter regulatory elements (e.g., derived from SV40, CMV,adenovirus and the like, such as a CMV enhancer/AdMLP promoterregulatory element or an SV40 enhancer/AdMLP promoter regulatoryelement) to drive high levels of transcription of the genes. Therecombinant expression vector also carries a DHFR gene, which allows forselection of CHO cells that have been transfected with the vector usingmethotrexate selection/amplification. The selected transformant hostcells are cultured to allow for expression of the antibody heavy andlight chains and intact antibody is recovered from the culture medium.Standard molecular biology techniques are used to prepare therecombinant expression vector, transfect the host cells, select fortransformants, culture the host cells and recover the antibody from theculture medium. For example, some antibodies can be isolated by affinitychromatography with a Protein A or Protein G coupled matrix.

For antibodies that include an Fc domain, the antibody production systemmay produce antibodies in which the Fc region is glycosylated. Forexample, the Fc domain of IgG molecules is glycosylated at asparagine297 in the CH2 domain. This asparagine is the site for modification withbiantennary-type oligosaccharides. It has been demonstrated that thisglycosylation is required for effector functions mediated by Fcgreceptors and complement Clq (Burton and Woof, 1992, Adv. Immunol.51:1-84; Jefferis et al., 1998, Immunol. Rev. 163:59-76). In oneembodiment, the Fc domain is produced in a mammalian expression systemthat appropriately glycosylates the residue corresponding to asparagine297. The Fc domain can also include other eukaryotic post-translationalmodifications.

Antibodies can also be produced by a transgenic animal. For example,U.S. Pat. No. 5,849,992 describes a method of expressing an antibody inthe mammary gland of a transgenic mammal. A transgene is constructedthat includes a milk-specific promoter and nucleic acids encoding theantibody of interest and a signal sequence for secretion. The milkproduced by females of such transgenic mammals includes,secreted-therein, the antibody of interest. The antibody can be purifiedfrom the milk, or for some applications, used directly.

Characterization of Plasma Kallikrein Binding Proteins

IC₅₀ (Inhibitory Concentration 50%) and EC₅₀ (Effective Concentration50%).

Within a series or group of binding proteins, those having lower IC₅₀ orEC₅₀ values are considered more potent inhibitors of plasma kallikreinthan those binding proteins having higher IC₅₀ or EC₅₀ values. Exemplarybinding proteins have an IC₅₀ value of less than 800 nM, 400 nM, 100 nM,25 nM, 5 nM, or 1 nM, e.g., as measured in an in vitro assay forinhibition of plasma kallikrein activity when the plasma kallikrein isat 2 pM.

Plasma kallikrein binding proteins may also be characterized withreference to the activity of Factor XII and HMWK (high-molecular-weightkininogen) signaling events, e.g., the production of Factor XIIa and/orbradykinin.

The binding proteins can also be evaluated for selectivity toward plasmakallikrein. For example, a plasma kallikrein binding protein can beassayed for its potency toward plasma kallikrein and a panel ofkallikreins and an IC₅₀ value or EC₅₀ value can be determined for eachkallikrein. In one embodiment, a compound that demonstrates a low IC₅₀value or EC₅₀ value for the plasma kallikrein, and a higher IC₅₀ valueor EC₅₀ value, e.g., at least 2-, 5-, or 10-fold higher, for anotherkallikrein within the test panel is considered to be selective towardplasma kallikrein.

A pharmacokinetics study in rat, mice, or monkey can be performed withplasma kallikrein binding proteins for determining plasma kallikreinhalf-life in the serum. Likewise, the effect of the binding protein canbe assessed in vivo, e.g., in an animal model for a disease (e.g.,carrageenin-induced edema in rat hind paw (Winter et al. Proc Soc ExpBiol Med. 1962; 111:544-7)), for use as a therapeutic, for example, totreat a disease or condition described herein, e.g., a plasma kallikreinassociated disorder.

Pharmaceutical Compositions

Proteins (e.g., binding proteins) that bind to plasma kallikrein (e.g.,human plasma kallikrein and/or murine plasma kallikrein) and, e.g.,include at least one immunoglobin variable region can be used in methodsfor treating (or preventing) a plasma kallikrein associated disease orcondition. The binding proteins can be present in a composition, e.g., apharmaceutically acceptable composition or pharmaceutical composition,which includes a plasma kallikrein-binding protein, e.g., an antibodymolecule or other polypeptide or peptide identified as binding to plasmakallikrein, as described herein. The plasma kallikrein binding proteincan be formulated together with a pharmaceutically acceptable carrier.Pharmaceutical compositions include therapeutic compositions anddiagnostic compositions, e.g., compositions that include labeled plasmakallikrein binding proteins for in vivo imaging, and compositions thatinclude labeled plasma kallikrein binding proteins for treating (orpreventing) a plasma kallikrein associated disease.

A pharmaceutically acceptable carrier includes any and all solvents,dispersion media, coatings, antibacterial and antifungal agents,isotonic and absorption delaying agents, and the like that arephysiologically compatible. Preferably, the carrier is suitable forintravenous, intramuscular, subcutaneous, parenteral, spinal, orepidermal administration (e.g., by injection or infusion), althoughcarriers suitable for inhalation and intranasal administration are alsocontemplated. Depending on the route of administration, the plasmakallikrein binding protein may be coated in a material to protect thecompound from the action of acids and other natural conditions that mayinactivate the compound.

A pharmaceutically acceptable salt is a salt that retains the desiredbiological activity of the compound and does not impart any undesiredtoxicological effects (see e.g., Berge, S. M., et al., 1977, J. Pharm.Sci. 66:1-19). Examples of such salts include acid addition salts andbase addition salts. Acid addition salts include those derived fromnontoxic inorganic acids, such as hydrochloric, nitric, phosphoric,sulfuric, hydrobromic, hydroiodic, phosphorous, and the like, as well asfrom nontoxic organic acids such as aliphatic mono- and dicarboxylicacids, phenyl-substituted alkanoic acids, hydroxy alkanoic acids,aromatic acids, aliphatic and aromatic sulfonic acids, and the like.Base addition salts include those derived from alkaline earth metals,such as sodium, potassium, magnesium, calcium, and the like, as well asfrom nontoxic organic amines, such as N,N′-dibenzylethylenediamine,N-methylglucamine, chloroprocaine, choline, diethanolamine,ethylenediamine, procaine, and the like.

The compositions may be in a variety of forms. These include, forexample, liquid, semi-solid and solid dosage forms, such as liquidsolutions (e.g., injectable and infusible solutions), dispersions orsuspensions, tablets, pills, powders, liposomes and suppositories. Theform can depend on the intended mode of administration and therapeuticapplication. Many compositions are in the form of injectable orinfusible solutions, such as compositions similar to those used foradministration of humans with antibodies. An exemplary mode ofadministration is parenteral (e.g., intravenous, subcutaneous,intraperitoneal, intramuscular). In one embodiment, the plasmakallikrein binding protein is administered by intravenous infusion orinjection. In another preferred embodiment, the plasma kallikreinbinding protein is administered by intramuscular or subcutaneousinjection.

The phrases “parenteral administration” and “administered parenterally”as used herein means modes of administration other than enteral andtopical administration, usually by injection, and includes, withoutlimitation, intravenous, intramuscular, intraarterial, intrathecal,intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal,transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular,subarachnoid, intraspinal, epidural and intrasternal injection andinfusion.

The composition can be formulated as a solution, microemulsion,dispersion, liposome, or other ordered structure suitable to high drugconcentration. Sterile injectable solutions can be prepared byincorporating the binding protein in the required amount in anappropriate solvent with one or a combination of ingredients enumeratedabove, as required, followed by filtered sterilization. Generally,dispersions are prepared by incorporating the active compound into asterile vehicle that contains a basic dispersion medium and the requiredother ingredients from those enumerated above. In the case of sterilepowders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum drying and freeze-dryingthat yields a powder of the active ingredient plus any additionaldesired ingredient from a previously sterile-filtered solution thereof.The proper fluidity of a solution can be maintained, for example, by theuse of a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Prolonged absorption of injectable compositions can be brought about byincluding in the composition an agent that delays absorption, forexample, monostearate salts and gelatin.

A plasma kallikrein binding protein can be administered by a variety ofmethods, although for many applications, the preferred route/mode ofadministration is intravenous injection or infusion. For example, fortherapeutic applications, the plasma kallikrein binding protein can beadministered by intravenous infusion at a rate of less than 30, 20, 10,5, or 1 mg/min to reach a dose of about 1 to 100 mg/m² or 7 to 25 mg/m².The route and/or mode of administration will vary depending upon thedesired results. In certain embodiments, the active compound may beprepared with a carrier that will protect the compound against rapidrelease, such as a controlled release formulation, including implants,and microencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Manymethods for the preparation of such formulations are available. See,e.g., Sustained and Controlled Release Drug Delivery Systems, J. R.Robinson, ed., 1978, Marcel Dekker, Inc., New York.

Pharmaceutical compositions can be administered with medical devices.For example, in one embodiment, a pharmaceutical composition disclosedherein can be administered with a device, e.g., a needleless hypodermicinjection device, a pump, or implant.

In certain embodiments, a plasma kallikrein binding protein can beformulated to ensure proper distribution in vivo. For example, theblood-brain barrier (BBB) excludes many highly hydrophilic compounds. Toensure that the therapeutic compounds disclosed herein cross the BBB (ifdesired), they can be formulated, for example, in liposomes. For methodsof manufacturing liposomes, see, e.g., U.S. Pat. Nos. 4,522,811;5,374,548; and 5,399,331. The liposomes may comprise one or moremoieties that are selectively transported into specific cells or organs,thus enhance targeted drug delivery (see, e.g., V. V. Ranade, 1989, J.Clin. Pharmacol. 29:685).

Dosage regimens are adjusted to provide the optimum desired response(e.g., a therapeutic response). For example, a single bolus may beadministered, several divided doses may be administered over time or thedose may be proportionally reduced or increased as indicated by theexigencies of the therapeutic situation. It is especially advantageousto formulate parenteral compositions in dosage unit form for ease ofadministration and uniformity of dosage. Dosage unit form as used hereinrefers to physically discrete units suited as unitary dosages for thesubjects to be treated; each unit contains a predetermined quantity ofactive compound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms can be dictated by and directly dependent on(a) the unique characteristics of the active compound and the particulartherapeutic effect to be achieved, and (b) the limitations inherent inthe art of compounding such an active compound for the treatment ofsensitivity in individuals.

An exemplary, non-limiting range for a therapeutically orprophylactically effective amount of a binding protein (e.g., anantibody) disclosed herein is 0.1-20 mg/kg, more preferably 1-10 mg/kg.An anti-plasma kallikrein antibody can be administered, e.g., byintravenous infusion, e.g., at a rate of less than 30, 20, 10, 5, or 1mg/min to reach a dose of about 1 to 100 mg/m² or about 5 to 30 mg/m².For binding proteins smaller in molecular weight than an antibody,appropriate amounts can be proportionally less. Dosage values may varywith the type and severity of the condition to be alleviated. For aparticular subject, specific dosage regimens can be adjusted over timeaccording to the individual need and the professional judgment of theperson administering or supervising the administration of thecompositions.

The pharmaceutical compositions disclosed herein may include a“therapeutically effective amount” or a “prophylactically effectiveamount” of a plasma kallikrein binding protein disclosed herein. A“therapeutically effective amount” refers to an amount effective, atdosages and for periods of time necessary, to achieve the desiredtherapeutic result. A therapeutically effective amount of thecomposition may vary according to factors such as the disease state,age, sex, and weight of the individual, and the ability of the proteinto elicit a desired response in the individual. A therapeuticallyeffective amount is also one in which any toxic or detrimental effectsof the composition are outweighed by the therapeutically beneficialeffects.

A “therapeutically effective dosage” preferably modulates a measurableparameter, e.g., levels of circulating IgG antibodies by a statisticallysignificant degree or at least about 20%, more preferably by at leastabout 40%, even more preferably by at least about 60%, and still morepreferably by at least about 80% relative to untreated subjects. Theability of a compound to modulate a measurable parameter, e.g., adisease-associated parameter, can be evaluated in an animal model systempredictive of efficacy in human disorders and conditions, e.g., a plasmakallikrein associated disease. Alternatively, this property of acomposition can be evaluated by examining the ability of the compound tomodulate a parameter in vitro.

A “prophylactically effective amount” refers to an amount effective, atdosages and for periods of time necessary, to achieve the desiredprophylactic result. Typically, because a prophylactic dose is used insubjects prior to or at an earlier stage of disease, theprophylactically effective amount will be less than the therapeuticallyeffective amount.

Stabilization and Retention

In one embodiment, a plasma kallikrein binding protein is physicallyassociated with a moiety that improves its stabilization and/orretention in circulation, e.g., in blood, serum, lymph, or othertissues, e.g., by at least 1.5, 2, 5, 10, or 50 fold. For example, aplasma kallikrein binding protein can be associated with a polymer,e.g., a substantially non-antigenic polymer, such as polyalkylene oxidesor polyethylene oxides. Suitable polymers will vary substantially byweight. Polymers having molecular number average weights ranging fromabout 200 to about 35,000 (or about 1,000 to about 15,000, and 2,000 toabout 12,500) can be used. For example, a plasma kallikrein bindingprotein can be conjugated to a water soluble polymer, e.g., hydrophilicpolyvinyl polymers, e.g., polyvinylalcohol and polyvinylpyrrolidone. Anon-limiting list of such polymers include polyalkylene oxidehomopolymers such as polyethylene glycol (PEG) or polypropylene glycols,polyoxyethylenated polyols, copolymers thereof and block copolymersthereof, provided that the water solubility of the block copolymers ismaintained.

A plasma kallikrein binding protein can also be associated with acarrier protein, e.g., a serum albumin, such as a human serum albumin(see e.g., Smith, B J. et al., 2001 Bioconjug Chem 12(5): 750-756). Forexample, a translational fusion can be used to associate the carrierprotein with the plasma kallikrein binding protein.

A plasma kallikrein binding protein can also be modified as a HESylationderivative. Processes for HESylation of a plasma kallikrein bindingprotein utilize hydroxyethyl starch to modify the protein. HESylation ofa protein can extend the circulating half-life of the protein and alsoreduce renal clearance.

Kits

A plasma kallikrein binding protein described herein can be provided ina kit, e.g., as a component of a kit. For example, the kit includes (a)a plasma kallikrein binding protein, e.g., a composition (e.g., apharmaceutical composition) that includes a plasma kallikrein bindingprotein, and, optionally (b) informational material. The informationalmaterial can be descriptive, instructional, marketing or other materialthat relates to a method described herein and/or the use of a plasmakallikrein binding protein, e.g., for a method described herein.

The informational material of the kit is not limited in its form. In oneembodiment, the informational material can include information aboutproduction of the compound, molecular weight of the compound,concentration, date of expiration, batch or production site information,and so forth. In one embodiment, the informational material relates tousing the binding protein to treat, prevent, or diagnosis of disordersand conditions, e.g., a plasma kallikrein associated disease orcondition.

In one embodiment, the informational material can include instructionsto administer a plasma kallikrein binding protein in a suitable mannerto perform the methods described herein, e.g., in a suitable dose,dosage form, or mode of administration (e.g., a dose, dosage form, ormode of administration described herein). In another embodiment, theinformational material can include instructions to administer a plasmakallikrein binding protein to a suitable subject, e.g., a human, e.g., ahuman having, or at risk for, a disorder or condition described herein,e.g., a plasma kallikrein associated disease or condition. For example,the material can include instructions to administer a plasma kallikreinbinding protein to a patient with a disorder or condition describedherein, e.g., a plasma kallikrein associated disease. The informationalmaterial of the kits is not limited in its form. In many cases, theinformational material, e.g., instructions, is provided in print but mayalso be in other formats, such as computer readable material.

A plasma kallikrein binding protein can be provided in any form, e.g.,liquid, dried or lyophilized form. It is preferred that a plasmakallikrein binding protein be substantially pure and/or sterile. When aplasma kallikrein binding protein is provided in a liquid solution, theliquid solution preferably is an aqueous solution, with a sterileaqueous solution being preferred. When a plasma kallikrein bindingprotein is provided as a dried form, reconstitution generally is by theaddition of a suitable solvent. The solvent, e.g., sterile water orbuffer, can optionally be provided in the kit.

The kit can include one or more containers for the compositioncontaining a plasma kallikrein binding protein. In some embodiments, thekit contains separate containers, dividers or compartments for thecomposition and informational material. For example, the composition canbe contained in a bottle, vial, or syringe, and the informationalmaterial can be contained in association with the container. In otherembodiments, the separate elements of the kit are contained within asingle, undivided container. For example, the composition is containedin a bottle, vial or syringe that has attached thereto the informationalmaterial in the form of a label. In some embodiments, the kit includes aplurality (e.g., a pack) of individual containers, each containing oneor more unit dosage forms (e.g., a dosage form described herein) of aplasma kallikrein binding protein. For example, the kit includes aplurality of syringes, ampules, foil packets, or blister packs, eachcontaining a single unit dose of a plasma kallikrein binding protein.The containers of the kits can be air tight, waterproof (e.g.,impermeable to changes in moisture or evaporation), and/or light-tight.

The kit optionally includes a device suitable for administration of thecomposition, e.g., a syringe, inhalant, dropper (e.g., eye dropper),swab (e.g., a cotton swab or wooden swab), or any such delivery device.In one embodiment, the device is an implantable device that dispensesmetered doses of the binding protein. The disclosure also features amethod of providing a kit, e.g., by combining components describedherein.

Treatments

Proteins that bind to plasma kallikrein, e.g., as described herein, havetherapeutic and prophylactic utilities, particularly in human subjects.These binding proteins are administered to a subject to treat, prevent,and/or diagnose a variety of disorders and conditions, including e.g., aplasma kallikrein associated disease, or even to cells in culture, e.g.,in vitro or ex vivo. For example, these binding proteins can be used tomodify the effects of plasma kallikrein released from cells in culture(Lilla et al., J Biol Chem. 284(20):13792-13803 (2009)). Treatingincludes administering an amount effective to alleviate, relieve, alter,remedy, ameliorate, improve or affect the disorder, the symptoms of thedisorder or the predisposition toward the disorder. The treatment mayalso delay onset, e.g., prevent onset, or prevent deterioration of adisease or condition.

As used herein, an amount of a target-binding agent effective to preventa disorder, or a prophylactically effective amount of the binding agentrefers to an amount of a target binding agent, e.g., an plasmakallikrein binding protein, e.g., an anti-plasma kallikrein antibodydescribed herein, which is effective, upon single- or multiple-doseadministration to the subject, for preventing or delaying the occurrenceof the onset or recurrence of a disorder, e.g., a disorder describedherein, e.g., a plasma kallikrein associated disease.

Methods of administering plasma kallikrein binding proteins and otheragents are also described in “Pharmaceutical Compositions.” Suitabledosages of the molecules used can depend on the age and weight of thesubject and the particular drug used. The binding proteins can be usedas competitive agents to inhibit, reduce an undesirable interaction,e.g., between plasma kallikrein and its substrate (e.g., Factor XII orHMWK). The dose of the plasma kallikrein binding protein can be theamount sufficient to block 90%, 95%, 99%, or 99.9% of the activity ofplasma kallikrein in the patient, especially at the site of disease.Depending on the disease, this may require 0.1, 1.0, 3.0, 6.0, or 10.0mg/Kg. For an IgG having a molecular mass of 150,000 g/mole (two bindingsites), these doses correspond to approximately 18 nM, 180 nM, 540 nM,1.08 μM, and 1.8 μM of binding sites for a 5 L blood volume.

In one embodiment, the plasma kallikrein binding proteins are used toinhibit an activity (e.g., inhibit at least one activity of plasmakallikrein, e.g., reduce Factor XIIa and/or bradykinin production) ofplasma kallikrein, e.g., in vivo. The binding proteins can be used bythemselves or conjugated to an agent, e.g., a cytotoxic drug, cytotoxinenzyme, or radioisotope. This method includes: administering the bindingprotein alone or attached to an agent (e.g., a cytotoxic drug), to asubject requiring such treatment. For example, plasma kallikrein bindingproteins that do not substantially inhibit plasma kallikrein may be usedto deliver nanoparticles containing agents, such as toxins, to plasmakallikrein associated cells or tissues, e.g., to treat a plasmakallikrein-associate disorder.

Because the plasma kallikrein binding proteins recognize plasmakallikrein expressing cells and can bind to cells that are associatedwith (e.g., in proximity of or intermingled with) a plasma kallikreinassociated disorder or condition, plasma kallikrein binding proteins canbe used to inhibit an activity (e.g., inhibit at least one activity ofplasma kallikrein, e.g., reduce Factor XIIa and/or bradykininproduction) any such cells and inhibit the plasma kallikrein associateddisease. Reducing plasma kallikrein activity can indirectly inhibitcells which may be dependent on the plasma kallikrein activity for thedevelopment and/or progression of a plasma kallikrein-associateddisorder.

The binding proteins may be used to deliver an agent (e.g., any of avariety of cytotoxic and therapeutic drugs) to cells and tissues whereplasma kallikrein is present. Exemplary agents include a compoundemitting radiation, molecules of plants, fungal, or bacterial origin,biological proteins, and mixtures thereof. The cytotoxic drugs can beintracellularly acting cytotoxic drugs, such as toxins short rangeradiation emitters, e.g., short range, high energy α-emitters.

To target plasma kallikrein expressing cells, a prodrug system can beused. For example, a first binding protein is conjugated with a prodrugwhich is activated only when in close proximity with a prodrugactivator. The prodrug activator is conjugated with a second bindingprotein, preferably one which binds to a non competing site on thetarget molecule. Whether two binding proteins bind to competing or noncompeting binding sites can be determined by conventional competitivebinding assays. Exemplary drug prodrug pairs are described in Blakely etal., (1996) Cancer Research, 56:3287 3292.

The plasma kallikrein binding proteins can be used directly in vivo toeliminate antigen-expressing cells via natural complement-dependentcytotoxicity (CDC) or antibody dependent cellular cytotoxicity (ADCC).The binding proteins described herein can include complement bindingeffector domain, such as the Fc portions from IgG1, -2, or -3 orcorresponding portions of IgM which bind complement. In one embodiment,a population of target cells is ex vivo treated with a binding agentdescribed herein and appropriate effector cells. The treatment can besupplemented by the addition of complement or serum containingcomplement. Further, phagocytosis of target cells coated with a bindingprotein described herein can be improved by binding of complementproteins. In another embodiment target, cells coated with the bindingprotein which includes a complement binding effector domain are lysed bycomplement.

Methods of administering plasma kallikrein binding proteins aredescribed in “Pharmaceutical Compositions.” Suitable dosages of themolecules used will depend on the age and weight of the subject and theparticular drug used. The binding proteins can be used as competitiveagents to inhibit or reduce an undesirable interaction, e.g., between anatural or pathological agent and the plasma kallikrein.

The plasma kallikrein binding protein can be used to deliver macro andmicromolecules, e.g., a gene into the cell for gene therapy purposesinto the endothelium or epithelium and target only those tissuesexpressing the plasma kallikrein. The binding proteins may be used todeliver a variety of cytotoxic drugs including therapeutic drugs, acompound emitting radiation, molecules of plants, fungal, or bacterialorigin, biological proteins, and mixtures thereof. The cytotoxic drugscan be intracellularly acting cytotoxic drugs, such as short rangeradiation emitters, including, for example, short range, high energy αemitters, as described herein.

In the case of polypeptide toxins, recombinant nucleic acid techniquescan be used to construct a nucleic acid that encodes the binding protein(e.g., antibody or antigen-binding fragment thereof) and the cytotoxin(or a polypeptide component thereof) as translational fusions. Therecombinant nucleic acid is then expressed, e.g., in cells and theencoded fusion polypeptide isolated.

Alternatively, the plasma kallikrein binding protein can be coupled tohigh energy radiation emitters, for example, a radioisotope, such as¹³¹I, a γ-emitter, which, when localized at a site, results in a killingof several cell diameters. See, e.g., S. E. Order, “Analysis, Results,and Future Prospective of the Therapeutic Use of Radiolabeled Antibodyin Cancer Therapy”, Monoclonal Antibodies for Cancer Detection andTherapy, R. W. Baldwin et al. (eds.), pp 303 316 (Academic Press 1985).Other suitable radioisotopes include a emitters, such as ²¹²Bi, ²¹³Bi,and ²¹¹At, and b emitters, such as ¹⁸⁶Re and ⁹⁰Y. Moreover, ¹⁷⁷Lu mayalso be used as both an imaging and cytotoxic agent.

Radioimmunotherapy (RIT) using antibodies labeled with ¹³¹I, ⁹⁰Y, and¹⁷⁷Lu is under intense clinical investigation. There are significantdifferences in the physical characteristics of these three nuclides andas a result, the choice of radionuclide is very critical in order todeliver maximum radiation dose to a tissue of interest. The higher betaenergy particles of ⁹⁰Y may be good for bulky tumors. The relatively lowenergy beta particles of ¹³¹I are ideal, but in vivo dehalogenation ofradioiodinated molecules is a major disadvantage for internalizingantibody. In contrast, ¹⁷⁷Lu has low energy beta particle with only0.2-0.3 mm range and delivers much lower radiation dose to bone marrowcompared to ⁹⁰Y. In addition, due to longer physical half-life (comparedto ⁹⁰Y), the residence times are higher. As a result, higher activities(more mCi amounts) of ¹⁷⁷Lu labeled agents can be administered withcomparatively less radiation dose to marrow. There have been severalclinical studies investigating the use of ¹⁷⁷Lu labeled antibodies inthe treatment of various cancers. (Mulligan T et al., 1995, Clin. Canc.Res. 1: 1447-1454; Meredith R F, et al., 1996, J. Nucl. Med.37:1491-1496; Alvarez R D, et al., 1997, Gynecol. Oncol. 65: 94-101).

Exemplary Diseases and Conditions

A plasma kallikrein binding protein described herein is useful to treat(or prevent) a disease or condition in which plasma kallikrein activityis implicated, e.g., a disease or condition described herein, or totreat (or prevent) one or more symptoms associated therewith. In someembodiments, the plasma kallikrein binding protein (e.g., plasmakallikrein binding IgG or Fab) inhibits plasma kallikrein activity.

Examples of such diseases and conditions which can be treated (orprevented) by a plasma kallikrein binding protein described hereininclude: rheumatoid arthritis, gout, intestinal bowel disease, oralmucositis, neuropathic pain, inflammatory pain, spinalstenosis-degenerative spine disease, arterial or venous thrombosis, postoperative ileus, aortic aneurysm, osteoarthritis, vasculitis, edema,hereditary angioedema, cerebral edema, pulmonary embolism, stroke,clotting induced by ventricular assistance devices or stents, headtrauma or peri-tumor brain edema, sepsis, acute middle cerebral artery(MCA) ischemic event (stroke), restenosis (e.g., after angioplasty),systemic lupus erythematosis nephritis, and burn injury. A plasmakallikrein binding protein described herein can also be used to promotewound healing. A plasma kallikrein binding protein described herein canalso be used as an oncology treatment by mechanisms that include, butare not limited to, blocking production of pro-angiogenic bradykinin.

A therapeutically effective amount of a plasma kallikrein bindingprotein can be administered to a subject having or suspected of having adisorder in which plasma kallikrein activity is implicated, therebytreating (e.g., ameliorating or improving a symptom or feature of adisorder, slowing, stabilizing and/or halting disease progression) thedisorder.

The plasma kallikrein binding protein can be administered in atherapeutically effective amount. A therapeutically effective amount ofa plasma kallikrein binding protein is the amount which is effective,upon single or multiple dose administration to a subject, in treating asubject, e.g., curing, alleviating, relieving or improving at least onesymptom of a disorder in a subject to a degree beyond that expected inthe absence of such treatment. A therapeutically effective amount of thecomposition may vary according to factors such as the disease state,age, sex, and weight of the individual, and the ability of the compoundto elicit a desired response in the individual. A therapeuticallyeffective amount is also one in which any toxic or detrimental effectsof the composition are outweighed by the therapeutically beneficialeffects. A therapeutically effective dosage preferably modulates ameasurable parameter, favorably, relative to untreated subjects. Theability of a compound to affect (e.g., inhibit) a measurable parametercan be evaluated in an animal model system predictive of efficacy in ahuman disorder.

Dosage regimens can be adjusted to provide the optimum desired response(e.g., a therapeutic response). For example, a single bolus may beadministered, several divided doses may be administered over time or thedose may be proportionally reduced or increased as indicated by theexigencies of the therapeutic situation. It is especially advantageousto formulate parenteral compositions in dosage unit form for ease ofadministration and uniformity of dosage. Dosage unit form as used hereinrefers to physically discrete units suited as unitary dosages for thesubjects to be treated; each unit contains a predetermined quantity ofactive compound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier.

Rheumatoid Arthritis

Rheumatoid arthritis (RA) is an autoimmune, chronic inflammatory diseasethat causes joint swelling and pain and normally results in jointdestruction. RA generally follows a relapsing/remitting course, with“flares” of disease activity interspersed with remissions of diseasesymptoms. RA is associated with a number of additional inflammatorydisorders, including Sjogren's syndrome (dry eyes and mouth caused byinflammation of tear and saliva glands), pleuritis (inflammation of thepleura that causes pain upon deep breath and coughing), rheumatoidnodules (nodular sites of inflammation that develop within the lungs),pericarditis (inflammation of the pericardium that causes pain whenlying down or leaning forward), Felty syndrome (splenomegaly andleucopenia observed in conjunction with RA, making the subject prone toinfection), and vasculitis (an inflammation of the blood vessels whichcan block blood flow). Plasma kallikrein has been implicated inrheumatoid arthritis.

Symptoms of active RA include fatigue, lack of appetite, low gradefever, muscle and joint aches, and stiffness. Muscle and joint stiffnessare usually most notable in the morning and after periods of inactivity.During flares, joints frequently become red, swollen, painful, andtender, generally as a consequence of synovitis.

Treatment for rheumatoid arthritis involves a combination ofmedications, rest, joint strengthening exercises, and joint protection.Two classes of medications are used in treating rheumatoid arthritis:anti-inflammatory “first-line drugs,” and “Disease-ModifyingAntirheumatic Drugs” (DMARDs). The first-line drugs include NSAIDS(e.g., aspirin, naproxen, ibuprofen, and etodolac) and cortisone(corticosteroids). DMARDs, such as gold (e.g., gold salts, goldthioglucose, gold thiomalate, oral gold), methotrexate, sulfasalazine,D-penicillamine, azathioprine, cyclophosphamide, chlorambucil, andcyclosporine, leflunomide, etanercept, infliximab, anakinra, andadalimumab, and hydroxychloroquine, promote disease remission andprevent progressive joint destruction, but they are notanti-inflammatory agents.

The disclosure provides methods of treating (e.g., ameliorating,stabilizing, or eliminating one or more symptoms or ameliorating orstabilizing the subject's score on a RA scale) rheumatoid arthritis byadministering a plasma kallikrein binding protein (e.g., atherapeutically effective amount of a plasma kallikrein binding protein)to a subject having or suspected of having RA. Additionally provided aremethods of treating RA by administering a plasma kallikrein bindingprotein (e.g., a therapeutically effective amount of a plasma kallikreinbinding protein) in combination with a second therapy, e.g., with atleast one anti-inflammatory “first line drug” (e.g., an NSAID and/orcortisone) and/or a DMARD. The disclosure also provides methods ofpreventing rheumatoid arthritis or a symptom thereof by administering aplasma kallikrein binding protein (e.g., a prophylactically effectiveamount of a plasma kallikrein binding protein) to a subject at risk ofdeveloping RA (e.g., a subject having a family member with RA or agenetic predisposition thereto).

Further provided are methods of treating (e.g., ameliorating,stabilizing, or eliminating one or more symptoms) rheumatoid arthritisassociated disorders (Sjogren's syndrome, pleuritis, pulmonaryrheumatoid nodules, pericarditis, Felty syndrome, and vasculitis) byadministering a plasma kallikrein binding protein (e.g., atherapeutically effective amount of a plasma kallikrein binding protein)to a subject having or suspected of having RA.

Scales useful for assessing RA and symptoms of RA include, e.g., theRheumatoid Arthritis Severity Scale (RASS; Bardwell et al., (2002)Rheumatology 41(1):38-45), SF-36 Arthritis Specific Health Index (ASHI;Ware et al., (1999) Med. Care. 37(5 Suppl):MS40-50), Arthritis ImpactMeasurement Scales or Arthritis Impact Measurement Scales 2 (AIMS orAIMS2; Meenan et al. (1992) Arthritis Rheum. 35(1):1-10); the StanfordHealth Assessment Questionnaire (HAQ), HAQII, or modified HAQ (see,e.g., Pincus et al. (1983) Arthritis Rheum. 26(11):1346-53).

Guidance for the determination of the dosage that delivers atherapeutically effective amount of a plasma kallikrein binding proteinmay be obtained from animal models of rheumatoid arthritis, such ascollagen-induced arthritis (CIA), which is induced, typically inrodents, by immunization with autologous or heterologous type IIcollagen in adjuvant (Williams et al. Methods Mol Med. 98:207-16(2004)).

Gout

Gout is a condition that results from crystals of uric acid depositingin tissues of the body. Gout is characterized by an overload of uricacid in the body and recurring attacks of joint inflammation(arthritis). Chronic gout can lead to deposits of hard lumps of uricacid in and around the joints, decreased kidney function, and kidneystones. Gout is often related to an inherited abnormality in the body'sability to process uric acid. Uric acid is a breakdown product ofpurines, which are part of many foods. An abnormality in handling uricacid can cause attacks of painful arthritis (gout attack), kidneystones, and blockage of the kidney filtering tubules with uric acidcrystals, leading to kidney failure. Some patients may only developelevated blood uric acid levels (hyperuricemia) without having arthritisor kidney problems.

Symptoms of gout include, e.g., excruciating and unexpected pain,swelling, redness, warmth and stiffness in the affected foot or otherparts of the body, and low-grade fever.

Treatments for gout include, e.g., nonsteroidal anti-inflammatory drugs(NSAIDs), colchicine and oral glucocorticoids, intra-articularglucocorticoids administered via a joint injection, xanthine oxidaseinhibitors (e.g., allopurinol, febuxostat), uricosurics (e.g.,probenecid, EDTA), urate oxidases (e.g., pegloticase), sodiumbicarbonate, and low purine diet.

The disclosure provides methods of treating (e.g., ameliorating,stabilizing, or eliminating one or more symptoms or the worsening of)gout by administering a plasma kallikrein binding protein (e.g., atherapeutically effective amount of a plasma kallikrein binding protein)to a subject having or suspected of having gout. Additionally providedare methods of treating gout by administering a plasma kallikreinbinding protein (e.g., a therapeutically effective amount of akallikrein binding protein) in combination with a second therapy, e.g.,an NSAID, a colchicine, an oral glucocorticoid, an intra-articularglucocorticoid administered via a joint injection, a xanthine oxidaseinhibitor (e.g., allopurinol, febuxostat), a uricosuric (e.g.,probenecid, EDTA), a urate oxidase (e.g., pegloticase), sodiumbicarbonate, and/or low purine diet. The disclosure also providesmethods of preventing gout or a symptom thereof by administering aplasma kallikrein binding protein (e.g., a prophylactically effectiveamount of a plasma kallikrein binding protein) to a subject at risk ofdeveloping gout (e.g., a subject having a family member with gout or agenetic predisposition thereto).

Guidance for the determination of the dosage that delivers atherapeutically effective amount of a plasma kallikrein binding proteinmay be obtained from animal models of gout, see, e.g., Reginato andOlsen, Curr Opin Rheumatol. 19(2):134-45 (2007) and references citedtherein.

Intestinal Bowel Disease (IBD)

Inflammatory bowel disease (IBD) is a group of inflammatory conditionsof the large intestine and, in some cases, the small intestine. The mainforms of IBD are Crohn's disease and ulcerative colitis (UC). Accountingfor far fewer cases are other forms of IBD: collagenous colitis,lymphocytic colitis, ischaemic colitis, diversion colitis, Behçet'ssyndrome, infective colitis, and indeterminate colitis. The maindifference between Crohn's disease and UC is the location and nature ofthe inflammatory changes. Crohn's can affect any part of thegastrointestinal tract, from mouth to anus (skip lesions), although amajority of the cases start in the terminal ileum. Ulcerative colitis,in contrast, is restricted to the colon and the rectum. Microscopically,ulcerative colitis is restricted to the mucosa (epithelial lining of thegut), while Crohn's disease affects the whole bowel wall. Finally,Crohn's disease and ulcerative colitis present with extra-intestinalmanifestations (such as liver problems, arthritis, skin manifestationsand eye problems) in different proportions.

Symptoms of IBD include abdominal pain, vomiting, diarrhea,hematochezia, weight loss, weight gain and various associated complaintsor diseases (arthritis, pyoderma gangrenosum, primary sclerosingcholangitis). Diagnosis is generally by colonoscopy with biopsy ofpathological lesions. Rarely, a definitive diagnosis of neither Crohn'sdisease nor ulcerative colitis can be made because of idiosyncrases inthe presentation. In this case, a diagnosis of indeterminate colitis maybe made.

Treatment for IBD, depending on the level of severity, may requireimmunosuppression to control the symptoms. Immunosuppresives such asazathioprine, methotrexate, or 6-mercaptopurine can be used. Morecommonly, treatment of IBD requires a form of mesalamine. Often,steroids are used to control disease flares and were once acceptable asa maintenance drug. Biologicals, such as infliximab, have been used totreat patients with Crohn's disease or Ulcerative Colitis. Severe casesmay require surgery, such as bowel resection, strictureplasty or atemporary or permanent colostomy or ileostomy. Alternative medicinetreatments for IBD exist in various forms however such methodsconcentrate on controlling underlying pathology in order to avoidprolonged steroidal exposure or surgical excision. Usually the treatmentis started by administering drugs, such as prednisone, with highanti-inflammatory affects. Once the inflammation is successfullycontrolled, the patient is usually switched to a lighter drug, such asasacol—a mesalamine— to keep the disease in remission. If unsuccessful,a combination of the aforementioned immunosuppressant drugs with amesalamine (which may also have an anti-inflammatory effect) may or maynot be administered, depending on the patient.

The disclosure provides methods of treating (e.g., ameliorating,stabilizing, or eliminating one or more symptoms of) IBD byadministering a plasma kallikrein binding protein (e.g., atherapeutically effective amount of a plasma kallikrein binding protein)to a subject having or suspected of having IBD. Additionally providedare methods of treating IBD by administering a plasma kallikrein bindingprotein (e.g., a therapeutically effective amount of a kallikreinbinding protein) in combination with a second therapy, e.g., animmunosuppressive (e.g., azathioprine, methotrexate, 6-mercaptopurine),a mesalamine, a steroid, and/or infliximab. The disclosure also providesmethods of preventing IBD or a symptom thereof by administering a plasmakallikrein binding protein (e.g., a prophylactically effective amount ofa plasma kallikrein binding protein) to a subject at risk of developingIBD (e.g., a subject having a family member with IBD or a geneticpredisposition thereto).

Guidance for the determination of the dosage that delivers atherapeutically effective amount of a plasma kallikrein binding proteinmay be obtained from animal models of IBD, see, e.g., those described inU.S. Pat. No. 6,114,382, WO 2004/071186, and references cited therein.

Oral Mucositis

Oral mucositis is the painful inflammation and ulceration of the mucousmembranes in the mouth, usually as an adverse effect of chemotherapy andradiotherapy treatment for cancer.

Symptoms of oral mucositis include, e.g., ulcers, peripheral erythema,burning sensation accompanied by reddening, trouble speaking, eating, oreven opening the mouth, and dyseusia (alteration in taste perception).

Treatment for oral mucositis includes oral hygiene (salt mouthwash,GELCLAIR®, CAPHOSOL®, MUGARD®), palifermin (a human keratinocyte growthfactor), cytokines and other modifiers of inflammation (e.g., IL-1,IL-11, TGF-beta3), amino acid supplementation (e.g., glutamine),vitamins, colony-stimulating factors, cryotherapy, and laser therapy.

The disclosure provides methods of treating (e.g., ameliorating,reducing, or eliminating one or more symptoms, or stabilizing thesubject's score on a mucositis scale) oral mucositis by administering aplasma kallikrein binding protein (e.g., a therapeutically effectiveamount of a plasma kallikrein binding protein) to a subject having orsuspected of having oral mucositis. Additionally provided are methods oftreating oral mucositis by administering a plasma kallikrein bindingprotein (e.g., a therapeutically effective amount of a plasma kallikreinbinding protein) in combination with a second therapy, e.g., oralhygiene (salt mouthwash, GELCLAIR®, CAPHOSOL®, MUGARD®), palifermin (ahuman keratinocyte growth factor), a cytokine and/or a modifier ofinflammation (e.g., IL-1, IL-11, TGF-beta3), an amino acidsupplementation (e.g., glutamine), a vitamin, a colony-stimulatingfactor, cryotherapy, and/or laser therapy. The disclosure also providesmethods of preventing oral mucositis or a symptom thereof byadministering a plasma kallikrein binding protein (e.g., aprophylactically effective amount of a plasma kallikrein bindingprotein) to a subject at risk of developing oral mucositis (e.g., asubject that has undergone or is undergoing chemotherapy orradiotherapy).

Scales useful for assessing oral mucositis include the World HealthOrganization (WHO) Oral Toxicity score (Handbook for reporting resultsof cancer treatment. Geneva, Switzerland: World Health Organization;1979:15-22), National Cancer Institute Common Toxicity Criteria(NCI-CTC) for Oral Mucositis (National Cancer Institute Common ToxicityCriteria. Version 2.0, Jun. 1, 1999, Sonis et al., Cancer. 85:2103-2113(1999)), and Oral Mucositis Assessment Scale (OMAS).

Guidance for the determination of the dosage that delivers atherapeutically effective amount of a plasma kallikrein binding proteinmay be obtained from animal models of oral mucositis, such as an animalmodel of oral mucositis induced by conditioning regimen ofhaematopoietic stem cell transplantation (Chen et al., Zhonghua KouQiang Yi Xue Za Zhi. 42(11):672-6 (2007)).

Neuropathic Pain

Neuropathic pain is a complex, chronic pain state that usually isaccompanied by tissue injury. With neuropathic pain, the nerve fibersthemselves may be damaged, dysfunctional or injured. These damaged nervefibers send incorrect signals to other pain centers. The impact of nervefiber injury includes a change in nerve function both at the site ofinjury and areas around the injury.

Symptoms of neuropathic pain include, e.g., shooting and burning painand tingling and numbness.

Treatments for neuropathic pain include, e.g., medications (e.g.,non-steroidal anti-inflammatory drugs (NSAIDs) (e.g., ALEVE®, MOTRIN®,or morphine), anticonvulsant, and antidepressant drugs), and invasive orimplantable devices (e.g., electrical stimulation).

The disclosure provides methods of treating (e.g., ameliorating,reducing, or eliminating one or more symptoms of or stabilizing thesubject's score on a pain scale) neuropathic pain by administering aplasma kallikrein binding protein (e.g., a therapeutically effectiveamount of a plasma kallikrein binding protein) to a subject having orsuspected of having neuropathic pain. Additionally provided are methodsof treating neuropathic pain by administering a plasma kallikreinbinding protein (e.g., a therapeutically effective amount of a plasmakallikrein binding protein) in combination with a second therapy, e.g.,a nonsurgical treatment ((e.g., a non-steroidal anti-inflammatory drug(NSAID) (e.g., ALEVE®, MOTRIN®, or morphine), an anticonvulsant, and/oran antidepressant drug), and/or an invasive or implantable device (e.g.,electrical stimulation). The disclosure also provides methods ofpreventing neuropathic pain or a symptom thereof by administering aplasma kallikrein binding protein (e.g., a prophylactically effectiveamount of a plasma kallikrein binding protein) to a subject at risk ofdeveloping neuropathic pain (e.g., a subject that has experienced tissueinjury).

Scales useful for the assessment of neuropathic pain include, e.g.,Wong-Baker FACES Pain Rating Scale (Wong-Baker FACES Pain Rating ScaleFoundation), Visual analog scale (VAS) (Huskisson, J. Rheumatol. 9 (5):768-9 (1982)), McGill Pain Questionnaire (MPQ) (Melzack, Pain 1 (3):277-99 (1975)), Descriptor differential scale (DDS) (Gracely andKwilosz, Pain 35 (3): 279-88 (1988)), Faces Pain Scale—Revised (FPS-R)(Hicks et al., Pain 93 (2): 173-83 (2001)), Numerical 11 point box(BS-11) (Jensen et al., Clin J Pain 5 (2): 153-9 (1989)), Numeric RatingScale (NRS-11) (Hartrick et al., Pain Pract 3 (4): 310-6 (2003)),Dolorimeter Pain Index (DPI) (Hardy et al., (1952). Pain Sensations andReactions. Baltimore: The Williams & Wilkins Co.), and Brief PainInventory (BPI) (Cleeland and Ryan Ann. Acad. Med. Singap. 23 (2):129-38 (1994)).

Guidance for the determination of the dosage that delivers atherapeutically effective amount of a plasma kallikrein binding proteinmay be obtained from animal models of neuropathic pain, see, e.g., thosedescribed in Martin et al., Methods Mol Med. 84:233-42 (2003) andreferences cited therein.

Inflammatory Pain

Inflammatory pain is caused by an insult such as penetration wounds,burns, extreme cold, fractures, arthritis, autoimmune conditions,excessive stretching, infections and vasoconstriction to the integrityof tissues at a cellular level. During inflammation a complexneuro-immune interaction results in primary hyperalgesia, in which alarge range of inflammatory molecules including prostaglandins andbradykinin induce and maintain the altered nociceptor sensitivity.

Treatments for inflammatory pain include, e.g., non-steroidalanti-inflammatory drugs (NSAIDs) and corticosteroids.

The disclosure provides methods of treating (e.g., ameliorating,reducing, or eliminating one or more symptoms of) inflammatory pain byadministering a plasma kallikrein binding protein (e.g., atherapeutically effective amount of a plasma kallikrein binding protein)to a subject having or suspected of having inflammatory pain.Additionally provided are methods of treating inflammatory pain byadministering a plasma kallikrein binding protein (e.g., atherapeutically effective amount of a plasma kallikrein binding protein)in combination with a second therapy, e.g., a non-steroidalanti-inflammatory drug (NSAID) and/or a corticosteroid. The disclosurealso provides methods of preventing inflammatory pain or a symptomthereof by administering a plasma kallikrein binding protein (e.g., aprophylactically effective amount of a plasma kallikrein bindingprotein) to a subject at risk of developing inflammatory pain (e.g., asubject that has experienced an insult, e.g., such as a penetrationwound, a burn, extreme cold, a fracture, arthritis, an autoimmunecondition, excessive stretching, or infection).

Scales useful for the assessment of inflammatory pain include, e.g.,Wong-Baker FACES Pain Rating Scale (Wong-Baker FACES Pain Rating ScaleFoundation), Visual analog scale (VAS) (Huskisson, J. Rheumatol. 9 (5):768-9 (1982)), McGill Pain Questionnaire (MPQ) (Melzack, Pain 1 (3):277-99 (1975)), Descriptor differential scale (DDS) (Gracely andKwilosz, Pain 35 (3): 279-88 (1988)), Faces Pain Scale—Revised (FPS-R)(Hicks et al., Pain 93 (2): 173-83 (2001)), Numerical 11 point box(BS-11) (Jensen et al., Clin J Pain 5 (2): 153-9 (1989)), Numeric RatingScale (NRS-11) (Hartrick et al., Pain Pract 3 (4): 310-6 (2003)),Dolorimeter Pain Index (DPI) (Hardy et al., (1952). Pain Sensations andReactions. Baltimore: The Williams & Wilkins Co.), and Brief PainInventory (BPI) (Cleeland and Ryan Ann. Acad. Med. Singap. 23 (2):129-38 (1994)).

Guidance for the determination of the dosage that delivers atherapeutically effective amount of a plasma kallikrein binding proteinmay be obtained from animal models of inflammatory pain such as ananimal model of chronic inflammatory pain (Wilson et al., Eur J Pain.10(6):537-49 (2006)) and an inflammatory model of pain and hyperalgesia(Ren and Dubner, ILAR J. 40(3):111-118 (1999)).

Spinal Stenosis

Spinal stenosis is a medical condition in which the spinal canal narrowsand compresses the spinal cord and nerves. This is usually due to thecommon occurrence of spinal degeneration that occurs with aging. It canalso sometimes be caused by spinal disc herniation, osteoporosis or atumor. Spinal stenosis may affect the cervical, thoracic or lumbarspine. In some cases, it may be present in all three places in the samepatient.

Symptoms of spinal stenosis include, e.g., pain or cramping in the legs,radiating back and hip pain, pain in the neck and shoulders, loss ofbalance, and loss of bowel or bladder function (cauda equina syndrome).

Treatments for spinal stenosis include, e.g., nonsurgical treatments(e.g., physical therapy, non-steroidal anti-inflammatory drugs (NSAIDs)(e.g., aspirin, ibuprofen and indomethacin), analgesics (e.g.,acetaminophen), chondroitin sulfate, glucosamine, rest or restrictedactivity, back brace or corset, epidural steroid injections (e.g.,corticosteroid)), and surgery (e.g., decompressive laminectomy,laminotomy and fusion).

The disclosure provides methods of treating (e.g., ameliorating,reducing, or eliminating one or more symptoms of) spinal stenosis byadministering a plasma kallikrein binding protein (e.g., atherapeutically effective amount of a plasma kallikrein binding protein)to a subject having or suspected of having spinal stenosis. Additionallyprovided are methods of treating spinal stenosis by administering aplasma kallikrein binding protein (e.g., a therapeutically effectiveamount of a plasma kallikrein binding protein) in combination with asecond therapy, e.g., a nonsurgical treatment (e.g., physical therapyand/or a nonsteroidal anti-inflammatory drug (NSAID) (e.g., aspirin,ibuprofen or indomethacin), an analgesic (e.g., acetaminophen),chondroitin sulfate, glucosamine, rest or restricted activity, a backbrace or corset, an epidural steroid injection (e.g., corticosteroid),and/or surgery (e.g., decompressive laminectomy, laminotomy and/orfusion). The disclosure also provides methods of preventing spinalstenosis or a symptom thereof by administering a plasma kallikreinbinding protein (e.g., a prophylactically effective amount of a plasmakallikrein binding protein) to a subject at risk of developing spinalstenosis (e.g., a subject that has spinal degeneration).

Guidance for the determination of the dosage that delivers atherapeutically effective amount of a plasma kallikrein binding proteinmay be obtained from animal models of spinal stenosis, such as a modelof lumbar spinal stenosis (Sekiguchi et al., Spine 29, 1105-1111(2004)).

Arterial and Venous Thrombosis

Arterial thrombosis is the formation of a thrombus within an artery. Inmost cases, arterial thrombosis follows rupture of atheroma, and istherefore referred to as atherothrombosis.

Arterial thrombosis is associated with a number of disorders, includingstroke and myocardial infarction. In thrombotic stroke, a thrombus(blood clot) usually forms around atherosclerotic plaques. Sinceblockage of the artery is gradual, onset of symptomatic thromboticstrokes is slower. Thrombotic stroke can be divided into twocategories—large vessel disease and small vessel disease. The formeraffects vessels such as the internal carotids, vertebral and the circleof Willis. The latter can affect smaller vessels such as the branches ofthe circle of Willis. Myocardial infarction (MI) is caused by an infarct(death of tissue due to ischemia), often due to the obstruction of thecoronary artery by a thrombus. MI can quickly become fatal if emergencymedical treatment is not received promptly.

Venous thrombosis is a blood clot that forms within a vein. If a pieceof a blood clot formed in a vein breaks off, it can be transported tothe right side of the heart, and from there into the lungs. A piece ofthrombus that is transported in this way is an embolism and the processof forming a thrombus that becomes embolic is called a thromboembolism.An embolism that lodges in the lungs is a pulmonary embolism (PE). Apulmonary embolus is a very serious condition that can be fatal if notrecognized and treated promptly.

Superficial venous thromboses can cause discomfort but generally do notcause serious consequences, unlike the deep venous thromboses (DVTs)that form in the deep veins of the legs or in the pelvic veins. Systemicembolisms of venous origin can occur in patients with an atrial orventricular septal defect, through which an embolus may pass into thearterial system. Such an event is termed a paradoxical embolism.

Prevention of arterial and/or venous thrombosis includes medications(e.g., anticoagulants (e.g., heparin), aspirin, and vitamin E) andmechanical methods (e.g., mechanical leg pumps (pneumatic compressionstockings)).

The disclosure provides methods of treating (e.g., ameliorating,reducing, or eliminating one or more symptoms of) arterial and/or venousthrombosis by administering a plasma kallikrein binding protein (e.g., atherapeutically effective amount of a plasma kallikrein binding protein)to a subject having or suspected of having arterial and/or venousthrombosis. Additionally provided are methods of treating arterialand/or venous thrombosis by administering a plasma kallikrein bindingprotein (e.g., a therapeutically effective amount of a plasma kallikreinbinding protein) in combination with a second therapy, e.g., ananticoagulant (e.g., heparin), aspirin, and/or vitamin E and/or amechanical method (e.g., a mechanical leg pump (pneumatic compressionstockings). The disclosure also provides methods of preventing arterialand/or venous thrombosis or a symptom thereof by administering a plasmakallikrein binding protein (e.g., a prophylactically effective amount ofa plasma kallikrein binding protein) to a subject at risk of developingarterial and/or venous thrombosis (e.g., a subject that has experienceda stroke or myocardial infarction).

Guidance for the determination of the dosage that delivers atherapeutically effective amount of a plasma kallikrein binding proteinmay be obtained from animal models of arterial or venous thrombosis,such as a double-tuck model of arterial thrombosis (Gomez-Jorge et al.,J. Vasc. Inter. Rad. 9(4): 633-638 (1998), a model of venous thrombosisin rat with low flow conditions in the venous blood stream (Fredrich etal., Blood Coagul Fibrinolysis. 5(2):243-8 (1994)), and a canine modelfor venous thrombosis and spontaneous pulmonary embolism (Frisbiel,Spinal Cord 43, 635-639 (2005)).

Postoperative Ileus

Postoperative ileus is a temporary paralysis of a portion of theintestines typically after an abdominal surgery. Postoperative ileuscommonly occurs for 24 to 72 hours after abdominal surgery.

Symptoms of postoperative ileus include, e.g., moderate and diffuseabdominal discomfort, constipation, abdominal distension, nausea orvomiting, lack of bowel movement and/or flatulence, and excessivebelching.

Treatments for postoperative ileus include, e.g., nil per os (NPO or“Nothing by Mouth”) until peristaltic sound is heard from auscultationof the area where this portion lies, nasogastric suction, parenteralfeeds, and medications (e.g., lactulose and erythromycin).

The disclosure provides methods of treating (e.g., ameliorating,reducing, or eliminating one or more symptoms of) postoperative ileus byadministering a plasma kallikrein binding protein (e.g., atherapeutically effective amount of a plasma kallikrein binding protein)to a subject having or suspected of having postoperative ileus.Additionally provided are methods of treating postoperative ileus byadministering a plasma kallikrein binding protein (e.g., atherapeutically effective amount of a plasma kallikrein binding protein)in combination with a second therapy, e.g., nil per os, nasogastricsuction, parenteral feeds, and/or a medication (e.g., lactulose and/orerythromycin). The disclosure also provides methods of preventingpostoperative ileus or a symptom thereof by administering a plasmakallikrein binding protein (e.g., a prophylactically effective amount ofa plasma kallikrein binding protein) to a subject at risk of developingpostoperative ileus (e.g., a subject that has had abdominal surgery).

Guidance for the determination of the dosage that delivers atherapeutically effective amount of a plasma kallikrein binding proteinmay be obtained from animal models of postoperative ileus, such as amodel to investigate postoperative ileus with strain gauge transducersin awake rats (Huge et al. J Surg Res. 74(2):112-8 (1998)).

Aortic Aneurysm

An aortic aneurysm is a general term for any swelling (dilatation oraneurysm) of the aorta, usually representing an underlying weakness inthe wall of the aorta at that location. Types of aortic aneurysmsinclude aortic root aneurysm, thoracic aortic aneurysm, abdominal aorticaneurysm, and thoracoabdominal aortic aneurysm.

Most intact aortic aneurysms do not produce symptoms. As they enlarge,symptoms of aortic aneurysm include, e.g., anxiety or feeling of stress,nausea or vomiting, clammy skin, rapid heart rate, abdominal pain, backpain may develop, leg pain or numbness, erythema nodosum (leg lesionstypically found near the ankle region), and a hoarse voice as the leftrecurrent laryngeal nerve winding around the arch of the aorta isstretched. Once an aneurysm is ruptured, it can cause severe pain andmassive internal hemorrhage, and is fatal in the absence of prompttreatment.

Treatments for aortic aneurysm include, e.g., medications, surgicaltreatment and endovascular treatment. Smaller aneurysms that are not athigh risk for rupturing can be treated with drugs to treat high bloodpressure, such as beta-blockers; or doxycycline for matrixmetalloproteinase-9 inhibition. Surgical treatment typically involvesopening up of the dilated portion of the aorta and insertion of asynthetic (Dacron or Gore-tex) patch tube. Endovascular treatment, as aminimally invasive alternative to open surgery repair, involves theplacement of an endovascular stent via a percutaneous technique (usuallythrough the femoral arteries) into the diseased portion of the aorta.

The disclosure provides methods of treating (e.g., stabilizing,reducing, or eliminating one or more symptoms of) aortic aneurysm byadministering a plasma kallikrein binding protein (e.g., atherapeutically effective amount of a plasma kallikrein binding protein)to a subject having or suspected of having aortic aneurysm. Additionallyprovided are methods of treating aortic aneurysm by administering aplasma kallikrein binding protein (e.g., a therapeutically effectiveamount of a plasma kallikrein binding protein) in combination with asecond therapy, e.g., a medication (e.g., a drug to treat high bloodpressure (e.g., a beta-blocker) or doxycycline), surgery, and/or anendovascular treatment. The disclosure also provides methods ofpreventing aortic aneurysm or a symptom thereof by administering aplasma kallikrein binding protein (e.g., a prophylactically effectiveamount of a plasma kallikrein binding protein) to a subject at risk ofdeveloping aortic aneurysm (e.g., a subject that has high bloodpressure).

Guidance for the determination of the dosage that delivers atherapeutically effective amount of a plasma kallikrein binding proteinmay be obtained from an animal model of aortic aneurysm, e.g., a ratmodel of abdominal aortic aneurysm using a combination of intraluminalelastase infusion and extraluminal calcium chloride exposure (Tanaka etal. J Vasc Surg. 50(6):1423-32 (2009)).

Osteoarthritis

Osteoarthritis, also known as degenerative arthritis, is characterizedby the breakdown and eventual loss of the cartilage of one or morejoints. Osteoarthritis occurs when the cartilage that cushions the endsof bones in the joints deteriorates over time. The smooth surface of thecartilage becomes rough, causing irritation. If the cartilage wears downcompletely, the ends of the bones will be damaged. Osteoarthritiscommonly affects the hands, feet, spine, and large weight-bearingjoints, such as the hips and knees.

Symptoms of osteoarthritis include, e.g., pain, tenderness, stiffness,loss of flexibility, grating sensation, and bone spurs.

Treatments for osteoarthritis include, e.g., conservative measures(e.g., rest, weight reduction, physical and occupational therapy) andmedications (e.g., acetaminophen, pain-relieving creams applied to theskin over the joints (e.g., capsaicin, salycin, methyl salicylate, andmenthol), non-steroidal anti-inflammatory drugs (NSAIDs) (e.g., aspirin,ibuprofen, nabumetone and naproxen), and Cox-2 inhibitors.

The disclosure provides methods of treating (e.g., stabilizing,reducing, or eliminating one or more symptoms or stabilizing thesubject's score on an osteoarthritis scale) osteoarthritis byadministering a plasma kallikrein binding protein (e.g., atherapeutically effective amount of a plasma kallikrein binding protein)to a subject having or suspected of having osteoarthritis. Additionallyprovided are methods of treating osteoarthritis by administering aplasma kallikrein binding protein (e.g., a therapeutically effectiveamount of a plasma kallikrein binding protein) in combination with asecond therapy, e.g., a conservative measure (e.g., rest, weightreduction, physical and/or occupational therapy) and/or a medication(e.g., acetaminophen, a topical pain-relieving cream, an NSAID (e.g.,aspirin, ibuprofen, nabumetone, or naproxen), and/or a Cox-2 inhibitor.The disclosure also provides methods of preventing osteoarthritis or asymptom thereof by administering a plasma kallikrein binding protein(e.g., a prophylactically effective amount of a plasma kallikreinbinding protein) to a subject at risk of developing osteoarthritis(e.g., a subject that has had a joint injury).

Scales useful for the assessment of osteoarthritis include, e.g., theKnee Injury and Osteoarthritis Outcome Score (KOOS; Roos et al. (1998)J. Orthop. Sports Phys. Ther. 28(2):88-96), Western Ontario and McMasterUniversities Osteoarthrtis Index (WOMAC; Roos et al. (2003) Health Qual.Life Outcomes 1(1):17), and the 36-item Short Form General Health Scale(SF-36 GHS), as well as other assessment tools known in the art.

Guidance for the determination of the dosage that delivers atherapeutically effective amount of a plasma kallikrein binding proteinmay be obtained from an animal model of osteoarthritis, e.g., injectionof mono-iodoacetate (MIA) into the femorotibial joint of rodents whichpromotes loss of articular cartilage similar to that noted in humanosteoarthritis (Guzman et al. Toxicol Pathol. 31(6):619-24 (2003)), andtransection of the anterior cruciate ligament (ACL) in canines to induceosteoarthritis (Fife and Brandt J Clin Invest. 84(5): 1432-1439 (1989)).

Vasculitis

Vasculitis refers to a heterogeneous group of disorders that arecharacterized by inflammatory destruction of blood vessels. Botharteries and veins can be affected. Lymphangitis is sometimes considereda type of vasculitis. Vasculitis is primarily due to leukocyte migrationand resultant damage. Vasculitis can be classified by the underlyingcause, the location of the affected vessels, or the type or size of theblood vessels. Vasculitis is associated with a number of additionaldisorders and conditions, e.g., Kawasaki disease, Behçet's disease,Polyarteritis nodosa, Wegener's granulomatosis, Cryoglobulinemia,Takayasu's arteritis, Churg-Strauss syndrome, Giant cell arteritis(temporal arteritis), Henoch-Schönlein purpura, Rheumatic diseases(e.g., rheumatoid arthritis and systemic lupus erythematosus), cancer(e.g., lymphomas), infections (e.g., hepatitis C), exposure to chemicalsand drugs (e.g., amphetamines, cocaine, and anthrax vaccines whichcontain the Anthrax Protective Antigen as the primary ingredient).

Symptoms of vasculitis include, e.g., fever, weight loss, palpablepurpura, livedo reticularis, myalgia or myositis, arthralgia orarthritis, mononeuritis multiplex, headache, stroke, tinnitus, reducedvisual acuity, acute visual loss, myocardial infarction, hypertension,gangrene, nose bleeds, bloody cough, lung infiltrates, abdominal pain,bloody stool, perforations, and glomerulonephritis.

Treatments for vasculitis include, e.g., cortisone-related medications(e.g., prednisone) and immune suppression drugs (e.g.,cyclophosphamide).

The disclosure provides methods of treating (e.g., stabilizing,reducing, or eliminating one or more symptoms or stabilizing thesubject's score on a vasculitis scale) vasculitis by administering aplasma kallikrein binding protein (e.g., a therapeutically effectiveamount of a plasma kallikrein binding protein) to a subject having orsuspected of having vasculitis. Additionally provided are methods oftreating vasculitis by administering a plasma kallikrein binding protein(e.g., a therapeutically effective amount of a plasma kallikrein bindingprotein) in combination with a second therapy (e.g., a cortisone-relatedmedication (e.g., prednisone) and/or an immune suppression drug (e.g.,cyclophosphamide)). The disclosure also provides methods of preventingvasculitis or a symptom thereof by administering a plasma kallikreinbinding protein (e.g., a prophylactically effective amount of a plasmakallikrein binding protein) to a subject at risk of developingvasculitis (e.g., a subject that has had Kawasaki disease, Behçet'sdisease, Polyarteritis nodosa, Wegener's granulomatosis,Cryoglobulinemia, or Takayasu's arteritis, and so forth).

The disclosure also provides methods of treating (e.g., stabilizing,reducing, or eliminating one or more symptoms or stabilizing thesubject's score on a vasculitis scale) vasculitis associated withsystemic lupus erythematosis by administering a plasma kallikreinbinding protein (e.g., a therapeutically effective amount of a plasmakallikrein binding protein) to a subject having or suspected of havingvasculitis associated with systemic lupus erythematosis. Additionallyprovided are methods of treating vasculitis associated with systemiclupus erythematosis by administering a plasma kallikrein binding protein(e.g., a therapeutically effective amount of a plasma kallikrein bindingprotein) in combination with a second therapy, e.g., a cortisone-relatedmedication (e.g., prednisone) and/or an immune suppression drug (e.g.,cyclophosphamide).

Further provided are methods of treating (e.g., ameliorating,stabilizing, or eliminating one or more symptoms) a vasculitisassociated disorder (Kawasaki disease, Behçet's disease, Polyarteritisnodosa, Wegener's granulomatosis, Cryoglobulinemia, Takayasu'sarteritis, Churg-Strauss syndrome, Giant cell arteritis (temporalarteritis), Henoch-Schönlein purpura, Rheumatic diseases (e.g.,rheumatoid arthritis and systemic lupus erythematosus), cancer (e.g.,lymphomas), infections (e.g., hepatitis C), exposure to chemicals anddrugs (e.g., amphetamines, cocaine, and anthrax vaccines which containthe Anthrax Protective Antigen as the primary ingredient)) byadministering a plasma kallikrein binding protein (e.g., atherapeutically effective amount of a plasma kallikrein binding protein)to a subject having or suspected of having a vasculitis associateddisorder. The disclosure also provides methods of preventing avasculitis associated disorder or a symptom thereof by administering aplasma kallikrein binding protein (e.g., a prophylactically effectiveamount of a plasma kallikrein binding protein) to a subject at risk ofdeveloping a vasculitis associated disorder.

Scales useful for the assessment of osteoarthritis include, e.g.,Birmingham Vasculitis Activity score (BVAS) version 3 (Mukhtyar et al.Ann Rheum Dis. 68(12):1827-32 (2009)), as well as other assessment toolsknown in the art.

Guidance for the determination of the dosage that delivers atherapeutically effective amount of a plasma kallikrein binding proteinmay be obtained from an animal model of vasculitis, see e.g., thosedescribed in Katz et al., Clin Rev Allergy Immunol. 35(1-2):11-8 (2008)and references cited therein.

Head Trauma

Head trauma refers to trauma to the head, which may or may not includeinjury to the brain. Types of head trauma include concussion, epiduralhematoma, subdural hematoma, cerebral contusion, and diffuse axonalinjury.

Symptoms of head trauma include, e.g., coma, confusion, drowsiness,personality change, seizures, nausea and vomiting, headache and a lucidinterval, during which a patient appears conscious only to deterioratelater, leaking cerebrospinal fluid, visible deformity or depression inthe head or face, an eye that cannot move or is deviated to one side canindicate that a broken facial bone is pinching a nerve that innervateseye muscles, wounds or bruises on the scalp or face, basilar skullfractures, a subcutaneous bleed over the mastoid, hemotympanum,cerebrospinal fluid rhinorrhea, and otorrhea.

Treatments for head trauma include, e.g., controlling elevatedintracranial pressure (e.g., sedation, paralytics, cerebrospinal fluiddiversion), decompressive craniectomy, barbiturate coma, hypertonicsaline, and hypothermia.

The disclosure provides methods of treating (e.g., stabilizing,reducing, or eliminating one or more symptoms or stabilizing thesubject's score on a head trauma scale) head trauma by administering aplasma kallikrein binding protein (e.g., a therapeutically effectiveamount of a plasma kallikrein binding protein) to a subject having orsuspected of having head trauma. Additionally provided are methods oftreating head trauma by administering a plasma kallikrein bindingprotein (e.g., a therapeutically effective amount of a plasma kallikreinbinding protein) in combination with a second therapy, e.g., controllingelevated intracranial pressure (e.g., sedation, a paralytic, and/orcerebrospinal fluid diversion), decompressive craniectomy, barbituratecoma, hypertonic saline, and/or hypothermia. The disclosure alsoprovides methods of preventing head trauma or a symptom thereof byadministering a plasma kallikrein binding protein (e.g., aprophylactically effective amount of a plasma kallikrein bindingprotein) to a subject at risk of developing head trauma (e.g., a subjectthat will be participating in a dangerous activity or contact sport).

Scales useful for assessing head trauma and symptoms of head traumainclude, e.g., the Glasgow Coma Scale (Teasdale and Jennett, Lancet 13;2(7872):81-4 (1974)), as well as other assessment tools known in theart.

Guidance for the determination of the dosage that delivers atherapeutically effective amount of a plasma kallikrein binding proteinmay be obtained from animal models of head trauma, see, e.g., thosedescribed in Cernak, NeuroRx. 2(3): 410-422 (2005) and references citedtherein.

Brain Edema

Brain edema (cerebral edema) is an excess accumulation of water in theintracellular and/or extracellular spaces of the brain. Types of brainedema include, e.g., vasogenic cerebral edema, cytotoxic cerebral edema,osmotic cerebral edema, and interstitial cerebral edema.

Vasogenic cerebral edema is due to a breakdown of tight endothelialjunctions which make up the blood-brain barrier (BBB). This allowsnormally excluded intravascular proteins and fluid to penetrate intocerebral parenchymal extracellular space. Once plasma constituents crossthe BBB, the edema spreads; this may be quite fast and widespread. Aswater enters white matter it moves extracellularly along fiber tractsand can also affect the gray matter. This type of edema is seen inresponse to trauma, tumors, focal inflammation, late stages of cerebralischemia and hypertensive encephalopathy. Some of the mechanismscontributing to BBB dysfunction are: physical disruption by arterialhypertension or trauma, tumor-facilitated release of vasoactive andendothelial destructive compounds (e.g., arachidonic acid, excitatoryneurotransmitters, eicosanoids, bradykinin, histamine and freeradicals). Some of the special subcategories of vasogenic edema include:hydrostatic cerebral edema, cerebral edema from brain cancer, highaltitude cerebral edema.

Cytotoxic cerebral edema is due to the derangement in cellularmetabolism resulting in inadequate functioning of the sodium andpotassium pump in the glial cell membrane. As a result there is cellularretention of sodium and water. Cytoxotic edema is seen with variousintoxications (dinitrophenol, triethyltin, hexachlorophene, isoniazid),in Reye's syndrome, severe hypothermia, early ischemia, encephalopathy,early stroke or hypoxia, cardiac arrest, pseudotumor cerebri, andcerebral toxins.

Osmotic cerebral edema occurs when plasma is diluted by excessive waterintake (or hyponatremia), syndrome of inappropriate antidiuretic hormonesecretion (SIADH), hemodialysis, or rapid reduction of blood glucose inhyperosmolar hyperglycemic state (HHS), formerly hyperosmolarnon-ketotic acidosis (HONK) and brain osmolality exceeds the serumosmolality creating an abnormal pressure.

Interstitial cerebral edema occurs in obstructive hydrocephalus. Thisform of edema is due to rupture of cerebral-spinal fluid (CSF)-brainbarrier resulting in trans-ependymal flow of CSF, which permits CSF topenetrate brain and spread in the extracellular space of white matter.

Symptoms of brain edema (e.g., peritumoral brain edema) include, e.g.,headache, loss of coordination (ataxia), weakness, and decreasing levelsof consciousness including disorientation, loss of memory,hallucinations, psychotic behavior, and coma.

Treatments for brain edema (e.g., peritumoral brain edema) include,e.g., medications (e.g. dexamethasone, mannitol, diuretics) and surgicaldecompression.

The disclosure provides methods of treating (e.g., stabilizing,reducing, or eliminating one or more symptoms of) brain edema (e.g.,peritumoral brain edema) by administering a plasma kallikrein bindingprotein (e.g., a therapeutically effective amount of a plasma kallikreinbinding protein) to a subject having or suspected of having brain edema(e.g., peritumoral brain edema). Additionally provided are methods oftreating brain edema (e.g., peritumoral brain edema) by administering aplasma kallikrein binding protein (e.g., a therapeutically effectiveamount of a plasma kallikrein binding protein) in combination with asecond therapy, e.g., a medication (e.g. dexamethasone, mannitol, and/ordiuretics) and/or surgical decompression. The disclosure also providesmethods of preventing brain edema or a symptom thereof by administeringa plasma kallikrein binding protein (e.g., a prophylactically effectiveamount of a plasma kallikrein binding protein) to a subject at risk ofdeveloping brain edema (e.g., a subject that has been diagnosed with abrain tumor).

Guidance for the determination of the dosage that delivers atherapeutically effective amount of a plasma kallikrein binding proteinmay be obtained from animal models of brain edema, e.g., a rat model ofcerebral embolism in which recirculation can be introduced in theischemic area (Koizumi et al., Jpn J Stroke 8: 1-8 (1986)).

Sepsis

Sepsis is a serious medical condition that is characterized by awhole-body inflammatory state and the presence of a known or suspectedinfection. This immunological response may be caused by microbes in theblood, urine, lungs, skin, or other tissues and can lead to widespreadactivation of acute-phase proteins, affecting the complement system andthe coagulation pathways, which then cause damage to the vasculature aswell as to the organs. Different levels of sepsis include systemicinflammatory response syndrome (SIRS), sepsis (SIRS in response to aconfirmed infectious process), severe sepsis (sepsis with organdysfunction, hypoperfusion, or hypotension), and septic shock (sepsiswith refractory arterial hypotension or hypoperfusion abnormalities inspite of adequate fluid resuscitation).

Symptoms of sepsis include, e.g., general symptoms related to theinfection, acute inflammation present throughout the entire body,hypothermia or fever, tachycardia, tachypnea or hypocapnia due tohyperventilation, leukopenia, leukocytosis, bandemia, and organ (e.g.,lung, brain, liver, kidney, and/or heart) dysfunction.

Treatments for sepsis include, e.g., antibiotics, vasopressor drugs,insulin, corticosteroids, drotrecogin alfa, surgical drainage ofinfected fluid collections, fluid replacement, and appropriate supportfor organ dysfunction (e.g., hemodialysis in kidney failure, mechanicalventilation in pulmonary dysfunction, transfusion of blood products, anddrug and fluid therapy for circulatory failure). Early Goal DirectedTherapy (EGDT), a systematic approach to resuscitation, can be used totreat severe sepsis and septic shock.

The disclosure provides methods of treating (e.g., stabilizing,reducing, or eliminating one or more symptoms or stabilizing thesubject's score on a sepsis scale) sepsis by administering a plasmakallikrein binding protein (e.g., a therapeutically effective amount ofa plasma kallikrein binding protein) to a subject having or suspected ofhaving sepsis. Additionally provided are methods of treating sepsis byadministering a plasma kallikrein binding protein (e.g., atherapeutically effective amount of a plasma kallikrein binding protein)in combination with a second therapy, e.g., an antibiotic, a vasopressordrug, insulin, a corticosteroid, drotrecogin alfa, surgical drainage ofinfected fluid collections, fluid replacement, an appropriate supportfor organ dysfunction (e.g., hemodialysis in kidney failure, mechanicalventilation in pulmonary dysfunction, transfusion of blood products,and/or drug and fluid therapy for circulatory failure), and/or an EarlyGoal Directed Therapy (EGDT). The disclosure also provides methods ofpreventing sepsis or a symptom thereof by administering a plasmakallikrein binding protein (e.g., a prophylactically effective amount ofa plasma kallikrein binding protein) to a subject at risk of developingsepsis (e.g., a subject that has been diagnosed as having an infection).

Scales useful for assessing sepsis and symptoms of sepsis include, e.g.,the Baltimore Sepsis Scale (Meek et al. J Burn Care Rehabil. 12(6):564-8(1991)) as well as other assessment tools known in the art.

Guidance for the determination of the dosage that delivers atherapeutically effective amount of a plasma kallikrein binding proteinmay be obtained from animal models of sepsis, see, e.g., those describedin U.S. Pat. No. 6,964,856, and Buras et al. Nat Rev Drug Discov.4(10):854-65 (2005) and references cited therein.

Acute Middle Cerebral Artery (MCA) Ischemic Event (Stroke)

An acute middle cerebral artery (MCA) ischemic event (stroke) is therapidly developing loss of brain function(s) due to disturbance in theblood supply to the brain due to ischemia (lack of glucose and oxygensupply) caused by thrombosis (e.g., venous thrombosis), embolism, orsystemic hypoperfusion. As a result, the affected area of the brain isunable to function, leading to inability to move one or more limbs onone side of the body, inability to understand or formulate speech, orinability to see one side of the visual field. A stroke is a medicalemergency and can cause permanent neurological damage, complications,and/or death.

Symptoms of acute middle cerebral artery (MCA) ischemic event (stroke)include, e.g., hemiplegia, decreased sensation and muscle weakness ofthe face, numbness, reduction in sensory or vibratory sensation, alteredsmell, taste, hearing or vision (total or partial), drooping of eyelid(ptosis) and weakness of ocular muscles, decreased reflexes, balanceproblems and nystagmus, altered breathing and heart rate, weakness insternocleidomastoid muscle with inability to turn head to one side,weakness in tongue (inability to protrude and/or move from side toside), aphasia, apraxia, visual field defect, memory deficits,hemineglect, disorganized thinking, confusion, hypersexual gestures,anosognosia, trouble walking, altered movement coordination, and vertigoand/or disequilibrium.

Treatment for acute middle cerebral artery (MCA) ischemic event (stroke)includes, e.g., thrombolysis (e.g., tissue plasminogen activator (tPA)),thrombectomy, angioplasty and stenting, therapeutic hypothermia, andmedications (e.g., aspirin, clopidogrel and dipyridamole).

The disclosure provides methods of treating (e.g., stabilizing,reducing, or eliminating one or more symptoms or stabilizing thesubject's score on a stroke scale) acute middle cerebral artery (MCA)ischemic event (stroke) by administering a plasma kallikrein bindingprotein (e.g., a therapeutically effective amount of a plasma kallikreinbinding protein) to a subject having or suspected of having acute middlecerebral artery (MCA) ischemic event (stroke). Additionally provided aremethods of treating acute middle cerebral artery (MCA) ischemic event(stroke) by administering a plasma kallikrein binding protein (e.g., atherapeutically effective amount of a plasma kallikrein binding protein)in combination with a second therapy, e.g., thrombolysis (e.g., tissueplasminogen activator (tPA)), thrombectomy, angioplasty and stenting,therapeutic hypothermia, and/or a medication (e.g., aspirin, clopidogreland dipyridamole). The disclosure also provides methods of preventingacute middle cerebral artery (MCA) ischemic event (stroke) or a symptomthereof by administering a plasma kallikrein binding protein (e.g., aprophylactically effective amount of a plasma kallikrein bindingprotein) to a subject at risk of developing acute middle cerebral artery(MCA) ischemic event (stroke) (e.g., a subject that has experiencedsystemic hypoperfusion).

Scales useful for assessing acute middle cerebral artery (MCA) ischemicevent (stroke) and symptoms of acute middle cerebral artery (MCA)ischemic event (stroke) include, e.g., Oxford Community Stroke Projectclassification (OCSP, also known as the Bamford or Oxfordclassification) (Bamford et al., Lancet 337 (8756): 1521-6 (1991)), andTOAST (Trial of Org 10172 in Acute Stroke Treatment) (Adams et al.,Stroke 24 (1): 35-41 (1993)).

Guidance for the determination of the dosage that delivers atherapeutically effective amount of a plasma kallikrein binding proteinmay be obtained from animal models of acute middle cerebral artery (MCA)ischemic event (stroke), see, e.g., those described in Beech et al.,Brain Res 895: 18-24 (2001), Buchan et al., Stroke 23 (2): 273-9 (1992),Carmichael, NeuroRx 2: 396-409 (2005), Chen et al., Stroke 17 (4):738-43 (1986), Dittmar et al., Stroke 34: 2252-7 (2003), Dittmar et al.,J Neurosci Methods 156: 50 (2006), Gerriets et al., J Neurosci Methods122: 201-11 (2003), Gerriets et al., Stroke 35: 2372-2377 (2004), Grahamet al., Comp Med 54: 486-496 (2004), Koizumi et al., Jpn J Stroke 8: 1-8(2004), Longa et al., Stroke 20 (1): 84-91 (1989), Mayzel-Oreg, MagnReson Med 51: 1232-8 (2004), Schmid-Elsaesser et al., Stroke 29 (10):2162-70 (1989), Tamura et al., J Cereb Blood Flow Metab 1: 53-60 (1981),Watson et al., Ann Neurol 17: 497-504 (1985), and Zhang et al., J CerebBlood Flow Metab 17: 123-35 (1997).

Restenosis

Restenosis is the reoccurrence of stenosis, a narrowing of a bloodvessel, leading to restricted blood flow. Restenosis usually pertains toan artery or other large blood vessel that has become narrowed, receivedtreatment to clear the blockage such as angioplasty, and subsequentlybecome renarrowed. It can be defined as a reduction in the circumferenceof the lumen of 50% or more, and had a high incidence rate (25-50%) inpatients who had undergone balloon angioplasty, with the majority ofpatients needing further angioplasty within 6 months.

Treatments for restenosis include, e.g., additional angioplasty ifrestenosis occurs without a stent or at either end of a stent, repeatedangioplasty and insertion of another stent inside the original ifrestenosis occurs within a stent, drug-eluted stents, brachytherapy, andintracoronary radiation.

The disclosure provides methods of treating (e.g., stabilizing,reducing, or eliminating one or more symptoms of) restenosis (e.g.,after angioplasty) by administering a plasma kallikrein binding protein(e.g., a therapeutically effective amount of a plasma kallikrein bindingprotein) to a subject having or suspected of having restenosis (e.g.,after angioplasty). Additionally provided are methods of treatingrestenosis (e.g., after angioplasty) by administering a plasmakallikrein binding protein (e.g., a therapeutically effective amount ofa plasma kallikrein binding protein) in combination with a secondtherapy, e.g., angioplasty if restenosis occurs without a stent or ateither end of a stent, repeated angioplasty and insertion of anotherstent inside the original if restenosis occurs within a stent, adrug-eluted stent, brachytherapy, and/or intracoronary radiation. Thedisclosure also provides methods of preventing restenosis or a symptomthereof by administering a plasma kallikrein binding protein (e.g., aprophylactically effective amount of a plasma kallikrein bindingprotein) to a subject at risk of developing restenosis (e.g., a subjectthat has had stenosis).

Guidance for the determination of the dosage that delivers atherapeutically effective amount of a plasma kallikrein binding proteinmay be obtained from animal models of restenosis, see, e.g., thosedescribed in U.S. Pat. Nos. 5,304,122 and 6,034,053, and Kantor et al.,Cardiovasc Radiat Med. 1(1):48-54 (1999), and references cited therein.

Systemic Lupus Erythematosus Nephritis

Systemic lupus erythematosus nephritis is an inflammation of the kidneycaused by systemic lupus erythematosus (SLE), a chronic autoimmuneconnective tissue disease. SLE can be associated with vasculitis whichare disorders characterized by inflammatory destruction of bloodvessels.

Symptoms of systemic lupus erythematosus nephritis include, e.g.,general symptoms of kidney disease, weight gain, high blood pressure,darker foamy urine, and swelling around the eyes, legs, ankles orfingers.

Treatments for systemic lupus erythematosus nephritis include, e.g.,steroid therapy (e.g., corticosteroids), chemotherapy (e.g.,cyclophosphamide, azathioprine, mycophenolate mofetil, or cyclosporine),and immunosuppressant agents (e.g., mycophenolate mofetil andintravenous cyclophosphamide).

The disclosure provides methods of treating (e.g., stabilizing,reducing, or eliminating one or more symptoms or stabilizing thesubject's score on a lupus scale) systemic lupus erythematosus nephritisby administering a plasma kallikrein binding protein (e.g., atherapeutically effective amount of a plasma kallikrein binding protein)to a subject having or suspected of having systemic lupus erythematosusnephritis. Additionally provided are methods of treating systemic lupuserythematosus nephritis by administering a plasma kallikrein bindingprotein (e.g., a therapeutically effective amount of a plasma kallikreinbinding protein) in combination with a second therapy, e.g., steroidtherapy (e.g., a corticosteroid), chemotherapy (e.g., cyclophosphamide,azathioprine, mycophenolate mofetil, and/or cyclosporine), and/or animmunosuppressant agent (e.g., mycophenolate mofetil and/or intravenouscyclophosphamide). The disclosure also provides methods of preventingsystemic lupus erythematosus nephritis or a symptom thereof byadministering a plasma kallikrein binding protein (e.g., aprophylactically effective amount of a plasma kallikrein bindingprotein) to a subject at risk of developing systemic lupus erythematosusnephritis (e.g., a subject that has been diagnosed with lupus or asubject having a family member with lupus or a genetic predispositionthereto).

Scales useful for assessing systemic lupus erythematosus nephritis andsymptoms of systemic lupus erythematosus nephritis include, e.g., WorldHealth Organization (WHO) classification based on the biopsy (Weening etal., J. Am. Soc. Nephrol. 15 (2): 241-50 (2004)) as well as otherassessment tools known in the art.

Guidance for the determination of the dosage that delivers atherapeutically effective amount of a plasma kallikrein binding proteinmay be obtained from animal models of systemic lupus erythematosusnephritis, see, e.g., those described in U.S. Pat. No. 7,265,261, Peng,Methods Mol Med. 102:227-72 (2004), and references cited therein.

Burn Injury and Wound Healing

A burn injury is a type of injury that may be caused by heat,electricity, chemicals, light, radiation, or friction. Muscle, bone,blood vessel, dermal and epidermal tissue can all be damaged withsubsequent pain due to profound injury to nerves. Depending on thelocation affected and the degree of severity, a burn victim mayexperience a wide number of potentially fatal complications includingshock, infection, electrolyte imbalance and respiratory distress. Inburn injuries, the damage to epidermis and dermal elements is the resultof several key insults which can be divided into initial (e.g., heatinjury, inflammatory mediator injury, ischemia induced injury) anddelayed insults. Excess heat causes rapid protein denaturation and celldamage. Much of the tissue damage, e.g., in the perfused subsurfaceburn, can be caused by toxic mediators of inflammation (e.g., oxidantsand/or proteases) which are activated with the burn. Consumption ofwound oxygen by neutrophils can lead to tissue hypoxia. Instant surfacevascular thrombosis occurs along with cell death from the heat insultand causes ischemia and further tissue damage. Delayed injury after theinitial heat and mediator damage includes, e.g., inflammation caused byneurotic tissue, bacteria on surface, caustic topical agents, andsurface exudate; and continued damage to viable cells and new tissuegrowth by excess wound proteolytic activity and oxidant release.

Treatments of burn injury include, e.g., intravenous fluids, dressings,pain management (e.g., analgesics (e.g., ibuprofen and acetaminophen),narcotics, and local anesthetics), inflammatory mediator inhibitors, andantibiotics.

The disclosure provides methods of treating (e.g., stabilizing,reducing, or eliminating one or more symptoms or stabilizing thesubject's score on a burn scale) a burn injury and/or promoting woundhealing by administering a plasma kallikrein binding protein (e.g., atherapeutically effective amount of a plasma kallikrein binding protein)to a subject having or suspected of having a burn injury. Additionallyprovided are methods of treating a burn injury by administering a plasmakallikrein binding protein (e.g., a therapeutically effective amount ofa plasma kallikrein binding protein) in combination with a secondtherapy, e.g., intravenous fluid, a dressing, pain management (e.g., ananalgesic (e.g., ibuprofen and acetaminophen), a narcotic, and a localanesthetic), an inflammatory mediator inhibitor, and an antibiotic. Thedisclosure also provides methods of preventing burn injuries or asymptom thereof by administering a plasma kallikrein binding protein(e.g., a prophylactically effective amount of a plasma kallikreinbinding protein) to a subject at risk of developing burn injuries (e.g.,a subject whose occupation creates a risk of a burn injury, e.g.,firefighter or cook).

Scales useful for assessing burns and symptoms of burns include, e.g.,burn scales by degrees, by thickness, and by total body surface area(TBSA) (Meek et al. J Burn Care Rehabil. 12(6):564-8 (1991)) as well asother assessment tools known in the art.

Guidance for the determination of the dosage that delivers atherapeutically effective amount of a plasma kallikrein binding proteinmay be obtained from animal models of burn, such as a porcine burn model(Singer and McClain, Methods Mol Med. 78:107-19 (2003), a sheep model ofthermal injury (Jonkam et al., Shock, 28:704-709 (2007)), a rabbit modelof thermal injury (Nwariaku et al., Burns, 22:324-327 (1996)), and amouse model of burn wounding (Stevenson et al., Methods Mol Med.78:95-105 (2003)).

Combination Therapies

A plasma kallikrein binding protein described herein, e.g., ananti-plasma kallikrein antibody, e.g., an anti-plasma kallikrein Fab orIgG, can be administered in combination with one or more of the othertherapies for treating a disease or condition associated with plasmakallikrein activity, e.g., a disease or condition described herein. Forexample, a plasma kallikrein binding protein can be used therapeuticallyor prophylactically with surgery, another anti-plasma kallikrein Fab orIgG (e.g., another Fab or IgG described herein), another plasmakallikrein inhibitor, a peptide inhibitor, or small molecule inhibitor.Examples of plasma kallikrein inhibitors that can be used in combinationtherapy with a plasma kallikrein binding protein described hereininclude plasma kallikrein inhibitors described in, e.g., WO 95/21601 orWO 2003/103475.

One or more plasma kallikrein inhibitors can be used in combination withone or more plasma kallikrein binding proteins described herein. Forexample, the combination can result in a lower dose of the inhibitorbeing needed, such that side effects are reduced.

A plasma kallikrein binding protein described herein can be administeredin combination with one or more current therapies for treating a plasmakallikrein associated disease or condition, including, but not limitedto the current therapies for treating the disorder, e.g., a currenttherapy for rheumatoid arthritis, gout, intestinal bowel disease, oralmucositis, neuropathic pain, inflammatory pain, spinalstenosis-degenerative spine disease, arterial or venous thrombosis, postoperative ileus, aortic aneurysm, osteoarthritis, vasculitis, headtrauma or peri-tumor brain edema, sepsis, acute middle cerebral artery(MCA) ischemic event (stroke), restenosis (e.g., after angioplasty),systemic lupus erythematosis nephritis, burn injury, or wound healing.For example, pKal inhibition is a novel mechanism of treating diseaseand therefore could provide effects that are synergistic or additivewith other therapeutics. For example, a protein described herein thatinhibits plasma kallikrein or that inhibits a downstream event of plasmakallikrein activity can also be used in combination with anothertreatment for a plasma kallikrein associated disease, such as surgery oradministration of a second agent, e.g., as described herein. Forexample, the second agent can include ecallantide, a C1 esteraseinhibitor (e.g., CINRYZE™), aprotinin (TRASYLOL®), a bradykinin B2receptor inhibitor (e.g., icatibant (FIRAZYR®)).

The term “combination” refers to the use of the two or more agents ortherapies to treat the same patient, wherein the use or action of theagents or therapies overlap in time. The agents or therapies can beadministered at the same time (e.g., as a single formulation that isadministered to a patient or as two separate formulations administeredconcurrently) or sequentially in any order. Sequential administrationsare administrations that are given at different times. The time betweenadministration of the one agent and another agent can be minutes, hours,days, or weeks. The use of a plasma kallikrein binding protein describedherein can also be used to reduce the dosage of another therapy, e.g.,to reduce the side effects associated with another agent that is beingadministered. Accordingly, a combination can include administering asecond agent at a dosage at least 10, 20, 30, or 50% lower than would beused in the absence of the plasma kallikrein binding protein.

The second agent or therapy can also be another agent for a plasmakallikrein associated therapy. Non-limiting examples of anothertreatment for a plasma kallikrein associated disease or conditioninclude, e.g., ecallantide, a C1 esterase inhibitor (e.g., CINRYZE™),aprotinin (TRASYLOL®), a bradykinin B2 receptor inhibitor (e.g.,icatibant (FIRAZYR®)) or a second binding protein described herein.

A combination therapy can include administering an agent that reducesthe side effects of other therapies. The agent can be an agent thatreduces the side effects of a plasma kallikrein associated diseasetreatment. For example, for inflammatory diseases, a pKal inhibitorcould be steroid sparring. Also, there could be synergism with aTNF-alpha inhibitor for treating inflammation or a VEGF blocker fortreating cancer and/or angiogenesis.

Diagnostic Uses

A protein that binds to plasma kallikrein described herein can have invitro and in vivo diagnostic utilities. A plasma kallikrein bindingprotein described herein (e.g., a protein that binds or binds andinhibits plasma kallikrein) can be used, e.g., for in vivo imaging,e.g., during a course of treatment for a disease or condition in whichplasma kallikrein is active, e.g., a disease or condition describedherein, or in diagnosing a disease or condition described herein.

In one aspect, the disclosure provides a diagnostic method for detectingthe presence of plasma kallikrein, in vitro or in vivo (e.g., in vivoimaging in a subject). The method can include localizing plasmakallikrein within a subject or within a sample from a subject. Withrespect to sample evaluation, the method can include, for example: (i)contacting a sample with plasma kallikrein binding protein; and (ii)detecting the location of the plasma kallikrein binding protein in thesample.

A plasma kallikrein binding protein can also be used to determine thequalitative or quantitative level of expression of plasma kallikrein ina sample. The method can also include contacting a reference sample(e.g., a control sample, e.g., a negative control) with the bindingprotein, and determining a corresponding assessment of the referencesample. A difference (e.g., increase), e.g., a statistically significantdifference, in the formation of the complex in the sample or subjectrelative to the control sample or subject can be indicative of thepresence of plasma kallikrein in the sample. In one embodiment, theplasma kallikrein binding protein does not cross react with anotherkallikrein protein, such as tissue kallikrein and/or with plasmaprekallikrein. E.g., the binding protein binds to another kallikreinprotein or to prekallikrein 5- to 10-fold less well (or even less well)than it binds to plasma kallikrein. For example, the binding protein canbind to plasma kallikrein with a KD of ˜10-50 pM, whereas it binds totissue kallikrein and/or prekallikrein at ˜10 nM.

The plasma kallikrein binding protein can be directly or indirectlylabeled with a detectable substance to facilitate detection of the boundor unbound antibody. Suitable detectable substances include variousenzymes, prosthetic groups, fluorescent materials, luminescent materialsand radioactive materials.

Complex formation between the plasma kallikrein binding protein andplasma kallikrein can be detected by evaluating the binding proteinbound to the plasma kallikrein or unbound binding protein. Conventionaldetection assays can be used, e.g., an enzyme-linked immunosorbentassays (ELISA), a radioimmunoassay (RIA) or tissue immunohistochemistry.Further to labeling the plasma kallikrein binding protein, the presenceof plasma kallikrein can be assayed in a sample by a competitionimmunoassay utilizing standards labeled with a detectable substance andan unlabeled plasma kallikrein binding protein. In one example of thisassay, the biological sample, the labeled standards, and the plasmakallikrein binding protein are combined and the amount of labeledstandard bound to the unlabeled binding protein is determined. Theamount of plasma kallikrein in the sample is inversely proportional tothe amount of labeled standard bound to the plasma kallikrein bindingprotein.

Fluorophore and chromophore labeled proteins can be prepared. Becauseantibodies and other proteins absorb light having wavelengths up toabout 310 nm, the fluorescent moieties should be selected to havesubstantial absorption at wavelengths above 310 nm and preferably above400 nm. A variety of suitable fluorescers and chromophores are describedby Stryer, 1968, Science 162:526 and Brand, L. et al., 1972, Annu. Rev.Biochem. 41:843-868. The proteins can be labeled with fluorescentchromophore groups by conventional procedures such as those disclosed inU.S. Pat. Nos. 3,940,475, 4,289,747, and 4,376,110. One group offluorescers having a number of the desirable properties described aboveis the xanthene dyes, which include the fluoresceins and rhodamines.Another group of fluorescent compounds are the naphthylamines. Oncelabeled with a fluorophore or chromophore, the protein can be used todetect the presence or localization of the plasma kallikrein in asample, e.g., using fluorescent microscopy (such as confocal ordeconvolution microscopy).

Histological Analysis.

Immunohistochemistry can be performed using the proteins describedherein. For example, in the case of an antibody, the antibody can besynthesized with a label (such as a purification or epitope tag), or canbe detectably labeled, e.g., by conjugating a label or label-bindinggroup. For example, a chelator can be attached to the antibody. Theantibody is then contacted to a histological preparation, e.g., a fixedsection of tissue that is on a microscope slide. After an incubation forbinding, the preparation is washed to remove unbound antibody. Thepreparation is then analyzed, e.g., using microscopy, to identify if theantibody bound to the preparation.

Of course, the antibody (or other polypeptide or peptide) can beunlabeled at the time of binding. After binding and washing, theantibody is labeled in order to render it detectable.

Protein Arrays.

The plasma kallikrein binding protein can also be immobilized on aprotein array. The protein array can be used as a diagnostic tool, e.g.,to screen medical samples (such as isolated cells, blood, sera,biopsies, and the like). Of course, the protein array can also includeother binding proteins, e.g., that bind to plasma kallikrein or to othertarget molecules.

Methods of producing polypeptide arrays are described, e.g., in De Wildtet al., 2000, Nat. Biotechnol. 18:989-994; Lueking et al., 1999, Anal.Biochem. 270:103-111; Ge, 2000, Nucleic Acids Res. 28, e3, I-VII;MacBeath and Schreiber, 2000, Science 289:1760-1763; WO 01/40803 and WO99/51773A1. Polypeptides for the array can be spotted at high speed,e.g., using commercially available robotic apparati, e.g., from GeneticMicroSystems or BioRobotics. The array substrate can be, for example,nitrocellulose, plastic, glass, e.g., surface-modified glass. The arraycan also include a porous matrix, e.g., acrylamide, agarose, or anotherpolymer.

For example, the array can be an array of antibodies, e.g., as describedin De Wildt, supra. Cells that produce the proteins can be grown on afilter in an arrayed format. Polypeptide production is induced, and theexpressed polypeptides are immobilized to the filter at the location ofthe cell. A protein array can be contacted with a labeled target todetermine the extent of binding of the target to each immobilizedpolypeptide. Information about the extent of binding at each address ofthe array can be stored as a profile, e.g., in a computer database. Theprotein array can be produced in replicates and used to compare bindingprofiles, e.g., of a target and a non-target.

FACS (Fluorescence Activated Cell Sorting).

The plasma kallikrein binding protein can be used to label cells, e.g.,cells in a sample (e.g., a patient sample). The binding protein is alsoattached (or attachable) to a fluorescent compound. The cells can thenbe sorted using fluorescence activated cell sorter (e.g., using a sorteravailable from Becton Dickinson Immunocytometry Systems, San JoseCalif.; see also U.S. Pat. Nos. 5,627,037; 5,030,002; and 5,137,809). Ascells pass through the sorter, a laser beam excites the fluorescentcompound while a detector counts cells that pass through and determineswhether a fluorescent compound is attached to the cell by detectingfluorescence. The amount of label bound to each cell can be quantifiedand analyzed to characterize the sample.

The sorter can also deflect the cell and separate cells bound by thebinding protein from those cells not bound by the binding protein. Theseparated cells can be cultured and/or characterized.

In Vivo Imaging.

Also featured is a method for detecting the presence of plasmakallikrein expressing tissues in vivo. The method includes (i)administering to a subject (e.g., a patient having, e.g., a plasmakallikrein associated disease or condition) an anti-plasma kallikreinantibody, conjugated to a detectable marker; (ii) exposing the subjectto a means for detecting said detectable marker to the plasma kallikreinexpressing tissues or cells. For example, the subject is imaged, e.g.,by NMR or other tomographic means.

Examples of labels useful for diagnostic imaging include radiolabelssuch as ¹³¹I, ¹¹¹In, ¹²³I, ^(99m)Tc, ³²P, ¹²⁵I, ³H, ¹⁴C, and ¹⁸⁸Rh,fluorescent labels such as fluorescein and rhodamine, nuclear magneticresonance active labels, positron emitting isotopes detectable by apositron emission tomography (“PET”) scanner, chemiluminescers such asluciferin, and enzymatic markers such as peroxidase or phosphatase.Short range radiation emitters, such as isotopes detectable by shortrange detector probes can also be employed. The protein can be labeledwith such reagents; for example, see Wensel and Meares, 1983,Radioimmunoimaging and Radioimmunotherapy, Elsevier, New York fortechniques relating to the radiolabeling of antibodies and D. Colcher etal., 1986, Meth. Enzymol. 121: 802-816.

The binding protein can be labeled with a radioactive isotope (such as¹⁴C, ³H, ¹²⁵I, ³²P, ¹³¹I). A radiolabeled binding protein can be usedfor diagnostic tests, e.g., an in vitro assay. The specific activity ofa isotopically-labeled binding protein depends upon the half life, theisotopic purity of the radioactive label, and how the label isincorporated into the antibody.

In the case of a radiolabeled binding protein, the binding protein isadministered to the patient, is localized to cells bearing the antigenwith which the binding protein reacts, and is detected or “imaged” invivo using known techniques such as radionuclear scanning using e.g., agamma camera or emission tomography. See e.g., A. R. Bradwell et al.,“Developments in Antibody Imaging”, Monoclonal Antibodies for CancerDetection and Therapy, R. W. Baldwin et al., (eds.), pp 65 85 (AcademicPress 1985). Alternatively, a positron emission transaxial tomographyscanner, such as designated Pet VI located at Brookhaven NationalLaboratory, can be used where the radiolabel emits positrons (e.g., ¹¹C,¹⁸F, ¹⁵O, and ¹³N).

MRI Contrast Agents.

Magnetic Resonance Imaging (MRI) uses NMR to visualize internal featuresof living subject, and is useful for prognosis, diagnosis, treatment,and surgery. MRI can be used without radioactive tracer compounds forobvious benefit. Some MRI techniques are summarized in EP-A-0 502 814.Generally, the differences related to relaxation time constants T1 andT2 of water protons in different environments are used to generate animage. However, these differences can be insufficient to provide sharphigh resolution images.

The differences in these relaxation time constants can be enhanced bycontrast agents. Examples of such contrast agents include a number ofmagnetic agents paramagnetic agents (which primarily alter T1) andferromagnetic or superparamagnetic (which primarily alter T2 response).Chelates (e.g., EDTA, DTPA and NTA chelates) can be used to attach (andreduce toxicity) of some paramagnetic substances (e.g., Fe⁺³, Mn⁺²,Gd⁺³). Other agents can be in the form of particles, e.g., less than 10mm to about 10 nM in diameter). Particles can have ferromagnetic,antiferromagnetic, or superparamagnetic properties. Particles caninclude, e.g., magnetite (Fe₃O₄), γ-Fe₂O₃, ferrites, and other magneticmineral compounds of transition elements. Magnetic particles mayinclude: one or more magnetic crystals with and without nonmagneticmaterial. The nonmagnetic material can include synthetic or naturalpolymers (such as sepharose, dextran, dextrin, starch and the like.

The plasma kallikrein binding protein can also be labeled with anindicating group containing of the NMR active ¹⁹F atom, or a pluralityof such atoms inasmuch as (i) substantially all of naturally abundantfluorine atoms are the ¹⁹F isotope and, thus, substantially all fluorinecontaining compounds are NMR active; (ii) many chemically activepolyfluorinated compounds such as trifluoracetic anhydride arecommercially available at relatively low cost; and (iii) manyfluorinated compounds have been found medically acceptable for use inhumans such as the perfluorinated polyethers utilized to carry oxygen ashemoglobin replacements. After permitting such time for incubation, awhole body MRI is carried out using an apparatus such as one of thosedescribed by Pykett, 1982, Sci. Am. 246:78 88 to locate and imagetissues expressing plasma kallikrein.

The following examples provide further illustration and are notlimiting.

EXAMPLES Example 1

We have discovered several antibody inhibitors and binders of plasmakallikrein (pKal). The most potent of these have been furthercharacterized and shown to have apparent inhibition constants(K_(i,app))<10 nM, to be specific pKal inhibitors with respect to othertested serine proteases, and to not bind prekallikrein. Amino acidsequences of the CDRs for the inhibitors and the binders are shown inTables 1 and 2, respectively.

TABLE 1CDR Amino Acid Sequences, ELISA Signal, and Apparent Inhibition Constant of AntibodyInhibitors of PKal (SEQ ID NOs: 8-223) HV- CDR1 Human (SEQ Human pKalLV-CDR1 LV-CDR2 LV-CDR3 ID HV-CDR2 HV-CDR3 Ini- pKal (Ki, (SEQ ID(SEQ ID (SEQ ID NOs: (SEQ ID (SEQ ID tial ELISA app NOs: NOs: NOs: 116-NOs: NOs: Name (T/B) nM) 8-43) 44-79) 80-115 151) 152-187) 188-223) M6-39.9 5.9 RASQSIRNYLN AASTLQS QQLSGYPHT FYYMV VIYPSGGITVYADSVKGDKWAVMPPYYYYAMDV D09 M7- 4.1 54 TGTNSDVGNYNLVS EVNKRPS CSYAGNRNFYV WYSMVSISPSGGLTNYADSVKG HTAARPFYYYYMDV B04 M7- 45.7 36 SGDKLGDKYAC QDSKRPSQAWDSSTGV WYLMI YIYPSGGFTYYADSVKG TEGPLSWGYGMDV E07 M8- 5.4 105SGDKLGNKYAY QDNNRPS QAWDSRTVV TYFML SIYPSGGNTVYADSVKG AASPVRNYYYYGMDVA09 M10- 39.2 <100  RASQSISVYLN GASNLQF QQTFSLFT FYNMN SISPSGGETNYADSVKGGGGAYRNNWWGGFDI F10 nM M10- 42.2 18 RASQSVSSSYLA GASSRAT QQYGSSPFT PYNMYSIRPSGGGTVYADSVKG GFIAARWYYFDY H05 M12- 48.5 5.2 SGDQLGDKYVG QDTKRPSQAWDTSTAG WYTMV RIYPSGGWTKYADSVKG EGLLWFGENAFDI D05 M27- 41.3 16SGDKLGDKYAC QDSKRPS QAWDSSTGV WYLMI YIYPSGGFTYYADSVKG TEGPLSWGYGMDV E05M28- 33.3 5.5 SGDQLGDKYVG QDTKRPS QAWDTSTAG WYTMV RIYPSGGWTKYADSVKGEGLLWFGENAFDI B11 M29- 47.5 0.7 SGNKLGDKYVA QDTKRPS QAWDSSIVI WYTMVYIYPSGGATFYADSVKG GSYDYIWGFYSDH D09 M29- 28.8 11 SGDNLGNKYNS QDTKRPSQAWDGNVV WYEMG SIYSSGGGTMYADSVKG NPQYSGYDRSLSDGAFDI E09 M35- 11.1 2.9RASQSVSSYLA DASNRAT QQRSNWPRGFT YYHMS VISPSGGSTKYADSVKGGGSSDYAWGSYRRPYYFDY G04 M38- 33.5 14 SGEKLGDKYVS EDSRRPS QAWDSSTAI YYMMVYIYSSGGHTVYADSVKG DLFLYDFWSKGAFDI F02 M41- 28.0 13 SGDKLGDKYTS QDIKRPSQAWDSPNARV HYRMS SIYPSGGRTVYADSVKG DKFEWRLLFRGIGNDAFDI A11 M73- 4.0<100  SGSSSNIGSNTVS NDHRRPS SAWDDSLNGVV RYEMY SISSSGGPTAYADSVKGGTPKWELLLRSIYIENAFDI D06 nM M76- 11.2 <100  RSSQSLSDDGNTYLD TLSYRASMQGTHWPPT FYAMH GIVPSGGRTHYADSVKG DSSGSPNPLFDY D01 nM M110- 2.4 <100 RSSLSLLHSNGYNYLD LSSTRAS MQPLETPPT YYEMD GISSSGGHTAYADSVKGERRSSSRARYYYGMDV C12 nM M137- 4.5 79 SGNNSNFGSNTVT SDSRRPS AAWDDSLNGVDYRMQ VIVPSGGNTMYADSVKG GGPGSSIAARRAPTGYYGMDV E12 M142- 29.9 0.2RASQPIDNYLN AASRLQS QQSYTVPYT AYSMI YIRPSGGRTTYADSVKG GGLLLWFRELKSNYFDYH08 M145- 6.2 1.1 RASQSVSSYLA DASNRAT QQRSNWPRGFT YYHMSVISPSGGSTKYADSVKG GGSSDYAWGSYRRPYYFDY D01 M145- 40.0 0.79 SGDKLGDKYTSQDIKRPS QAWDSPNARV HYRMS SIYPSGGRTVYADSVKG DKFEWRLLFRGIGNDAFDI D11 M146-49.6 2.2 RASGDIGNALG DASTLQS LQGYNYPRT RYIMH SISPSGGLTSYADSVKGEFENAYHYYYYGMDV E12 M152- 19. <100  RASQSISSYLS AASSLQS QQSISIPRT PYFMGGIGPSGGSTTYADSVKG EGPPYSSGWYRGLRQYHFDY A12 nM M160- 38.3 17 RASQGISSYLAAASTLQS QQLNSYPLT HYLMT YISPSGGHTIYADSVKG VARGIAARSRTSYFDY G12 M161-41.8 0.3 SGDKLGDKYVS QDTKRPS QAWDSSTYV DYAMK SISSSGGVTQYADSVKGEEDYSSSWYSRRFDYYYGMDV C11 M162- 11.4 4.8 RASQSISSWLA KASTLES QQYNTYWTHYIMM GIYSSGGITVYADSVKG RRTGIPRRDAFDI A04 X67- nd 2.1 RASQPIDNYLNAASRLQS QQSYTVPYT AYSMI YIRPSGGRTTYADSVKG GGLLLWSRELKSNYFDY B03 X67- nd0.7 RASQPIDNYLN AASRLQS QQSYTVPYT AYSMI YIRPSGGRTTYADSVKGGGLLLWFMELKSNYFDY C03 X67- nd 8.6 RASQPIDNYLN AASRLQS QQSYTVPYT AYSMIYIRPSGGRTTYADSVKG GGLLLWGRELKSNYFDY C09 X67- nd 0.1 RASQPIDNYLN AASRLQSQQSYTVPYT AYSMI YIRPSGGRTTYADSVKG GGLLLWNRELKSNYFDY D03 X67- nd 1.3RASQPIDNYLN AASRLQS QQSYTVPYT AYSMI YIRPSGGRTTYADSVKG GGLLLWDRELKSNYFDYE04 X67- nd 0.9 RASQPIDNYLN AASRLQS QQSYTVPYT AYSMI YIRPSGGRTTYADSVKGGGLLLWQRELKSNYFDY F01 X67- nd 1.3 RASQPIDNYLN AASRLQS QQSYTVPYT AYSMIYIRPSGGRTTYADSVKG GGLLLWTRELKSNYFDY F10 X67- nd 0.35 RASQPIDNYLN AASRLQSQQSYTVPYT AYSMI YIRPSGGRTTYADSVKG GGLLLWARELKSNYFDY G04 X67- nd 3.6RASQPIDNYLN AASRLQS QQSYTVPYT AYSMI YIRPSGGRTTYADSVKG GGLLLWERELKSNYFDYH04 X81- nd 0.2 RTSQFVNSNYLA GASSRAT QQSSRTPWT HYLMT YISPSGGHTIYADSVKGVARGIAARSRTSYFDY B01 Abbreviations used: “T/B” is the ELISA signalobtained using of the “target” (biotinylated plasma kallikrein) dividedby the ELISA signal of the “background” (streptavidin); both of whichwere coated on microtiter plates. “nd” is not determined. The symbol “q”refers to the amber suppressible stop codon (TAG), which is translatedas glutamine (Q) in strains of E. coli such as the TG1 cells that wereused to express the Fab fragments.

Amino acid sequences of light chain (LC) and heavy chain (HC) variabledomain of pKal antibody inhibitors are shown below (SEQ ID NOs:224-293numbered in order from top to bottom).

M6-D09 LCQDIQMTQSPS SLSASVGDRV TITCRASQSI RNYLNWYQQK PGKAPNLLIY AASTLQSGVP  60ARFSGSGSGT DFTLTISSLQ PEDFATYYCQ QLSGYPHTFG QGTKLEIK 108 M6-D09 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS FYYMVWVRQA PGKGLEWVSV IYPSGGITVY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARDK WAVMPPYYYY AMDVWGQGTT 120VTVSSASTKG PSVFPLAPSS KS 142 M7-B04 LCQSALTQPASV SGSPGQSITI SCTGTNSDVG NYNLVSWYQQ HPGEAPKLLI YEVNKRPSGV  60SNRFSGSKSG NTASLTISGL QAEDEADYLC CSYAGNRNFY VFGAGTKVTV L 111 M7-B04 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS WYSMVWVRQA PGKGLEWVSS ISPSGGLTNY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARHT AARPFYYYYM DVWGKGTTVT 120VSSASTKGPS VFPLAPSSKS 140 M7-E07 LCQSELTQPPSV SVSPGQTASI TCSGDKLGDK YACWYQQKPG QSPVLVIYQD SKRPSGIPER  60FSGSNSGNTA TLTISGTQAM DEADYYCQAW DSSTGVFGGG TKLTVL 106 M7-E07 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS WYLMIWVRQA PGKGLEWVSY IYPSGGFTYY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED MAVYYCARTE GPLSWGYGMD VWGQGTTVTV 120SSASTKGPSV FPLAPSSKS 139 M8-A09 LCQCELTQPPSE SVSPGQTANI TCSGDKLGNK YAYWYQQKPG QSPVLVIYQD NNRPSGIPER  60FSGSNSGNTA TLTISGTQAI DEANYYCQAW DSRTVVFGGG TKLTVL 106 M8-A09 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS TYFMLWVRQA PGKGLEWVSS IYPSGGNTVY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARAA SPVRNYYYYG MDVWGQGTTV 120TVSSASTKGP SVFPLAPSSK S 141 M10-F10 LCQDIQMTQSPS SLSASVGDRV TITCRASQSI SVYLNWYQHK PGKAPKLLIY GASNLQFGVP  60SRFSGSGYGT DFTLTISSLQ PEDFATYHCQ QTFSLFTFGG GTKVEIK 107 M10-F10 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS FYNMNWVRQA PGKGLEWVSS ISPSGGETNY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARGG GAYRNNWWGG FDIWGLGTMV 120TVSSASTKGP SVFPLAPSSK S 141 M10-H05 LCQDIQMTQSPG TLSLSPGERA TLSCRASQSV SSSYLAWYQQ KPGQAPRLLI YGASSRATGI  60PDRFSGSGSG TDFTLTISRL EPEDFAVYYC QQYGSSPFTF GPGTKVDIK 109 M10-H05 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS PYNMYWVRQA PGKGLEWVSS IRPSGGGTVY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAGGF IAARWYYFDY WGQGTLVTVS 120SASTKGPSVF PLAPSSKS 138 M12-D05 LCQSVLTQPPSV SVSPGQTATI TCSGDQLGDK YVGWYQQKPG QSPILVIYQD TKRPSGIPER  60FSGSNSGNTA TLTISGTHTV DEAHYYCQAW DTSTAGFGGG TKLTVL 106 M12-D05 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS WYTMVWVRQA PGKGLEWVSR IYPSGGWTKY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TATYYCAREG LLWFGENAFD IWGQGTMVTV 120SSASTKGPSV FPLAPSSKS 139 M27-E05 LCQSELTQPPSV SVSPGQTASI TCSGDKLGDK YACWYQQKPG QSPVLVIYQD SKRPSGIPER  60FSGSNSGNTA TLTISGTQAM DEADYYCQAW DSSTGVFGGG TKLTVL 106 M27-E05 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS WYLMIWVRQA PGKGLEWVSY IYPSGGFTYY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED MAVYYCARTE GPLSWGYGMD VWGQGTTVTV 120SSASTKGPSV FPLAPSSKS 139 M28-B11 LCQSVLTQPPSV SVSPGQTATI TCSGDQLGDK YVGWYQQKPG QSPILVIYQD TKRPSGIPER  60FSGSNSGNTA TLTISGTHTV DEAHYYCQAW DTSTAGFGGG TKLTVL 106 M28-B11 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS WYTMVWVRQA PGKGLEWVSR IYPSGGWTKY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TATYYCAREG LLWFGENAFD IWGQGTMVTV 120SSASTKGPSV FPLAPSSKS 139 M29-D09 LCQSALTQPPTV SVSPGQTARI TCSGNKLGDK YVAWYQQKPG QSPMLVIYQD TKRPSRVSER  60FSGSNSANTA TLSISGTQAL DEADYYCQAW DSSIVIFGGG TRLTVL 106 M29-D09 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS WYTMVWVRQA PGKGLEWVSY IYPSGGATFY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAMGS YDYIWGFYSD HWGQGTLVTV 120SSASTKGPSV FPLAPSSKS 139 M29-E09 LCQYELTQPPSV SVSPGQTATI TCSGDNLGNK YNSWYQQKPG QSPLLVIYQD TKRPSAIPER  60FSGSNSGNTA TLTISGTQAM DEADYYCQAW DGNVVFGGGT KLTVL 105 M29-E09 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS WYEMGWVRQA PGKGLEWVSS IYSSGGGTMY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARNP QYSGYDRSLS DGAFDIWGQG 120TMVTVSSAST KGPSVFPLAP SSKS 144 M35-G04 LCQDIQMTQSPA TLSLSPGERA TLSCRASQSV SSYLAWYQQK PGQAPRLLIY DASNRATGIP  60ARFSGSGSGT DFTLTISSLE PEDFAVYYCQ QRSNWPRGFT FGPGTKVDIK 110 M35-G04 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS YYHMSWVRQA PGKGLEWVSV ISPSGGSTKY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARGG SSDYAWGSYR RPYYFDYWGQ 120GTLVTVSSAS TKGPSVFPLA PSSKS 145 M38-F02 LCQSVLTQPPSV SVSPGQTASI TCSGEKLGDK YVSWYQQKPG QSPSLVICED SRRPSGIPER  60FSGSNSGNTA TLTISGAQPM DEADYYCQAW DSSTAIFGPG TKVTVL 106 M38-F02 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS YYMMVWVRQA PGKGLEWVSY IYSSGGHTVY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARDL FLYDFWSKGA FDIWGQGTMV 120TVSSASTKGP SVFPLAPSSK S 141 M41-A11 LCQSVLTQPPSV SVSPGQTASI TCSGDKLGDK YTSWYQQRPG QSPVLVIYQD IKRPSGIPER  60FSGSNSGNTA TLTISGTQAM DEADYYCQAW DSPNARVFGS GTKVTVL 107 M41-A11 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS HYRMSWVRQA PGKGLEWVSS IYPSGGRTVY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAKDK FEWRLLFRGI GNDAFDIWGQ 120GTMVTVSSAS TKGPSVFPLA PSSKS 145 M73-D06 LCQSELTQPPSA SETPGQRVTI SCSGSSSNIG SNTVSWFQQL PGSAPRLLIY NDHRRPSGVP  60DRFSGSKSGT SASLVISGLQ SQDEADYYCS AWDDSLNGVV FGGGTKLTVL 110 M73-D06 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS RYEMYWVRQA PGKGLEWVSS ISSSGGPTAY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAMYYCAKGT PKWELLLRSI YIENAFDIWG 120QGTMVTVSSA STKGPSVFPL APSSKS 146 M76-D01 LCQDIVMTQTPP SLPVNPGEPA SISCRSSQSL SDDGNTYLDW YLQRPGQSPQ LLIHTLSYRA  60SGVPDRFSGS GSGTDFTLKI SRVEAEDVGV YYCMQGTHWP PTFGQGTKVE IK 112 M76-D01 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS FYAMHWVRQA PGKGLEWVSG IVPSGGRTHY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCATDS SGSPNPLFDY WGQGTLVTVS 120SASTKGPSVF PLAPSPKS 138 M110-C12 LCQDIQMTQSPL SLSVTPGEPA SISCRSSLSL LHSNGYNYLD WYVQRPGQSP QLLMYLSSTR  60ASGVPDRFSG SGSGTDFTLE ISRVEAEDVG VYYCMQPLET PPTFGGGTKV EIK 113M110-C12 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS YYEMDWVRQA PGKGLEWVSG ISSSGGHTAY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TATYYCARER RSSSRARYYY GMDVWGQGTT 120VTVSSASTKG PSVFPLAPSS KS 142 M137-E12 LCQSVLIQPPSV SGIPGQRVTI SCSGNNSNFG SNTVTWYQQL PGTAPKLLIY SDSRRPSGVP  60DRFSGSRSDT SASLAISGLQ SEDEAEYHCA AWDDSLNGVF GGGTKLTVL 109 M137-E12 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS DYRMQWVRQA PGKGLEWVSV IVPSGGNTMY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARGG PGSSIAARRA PTGYYGMDVW 120GQGTTVTVSS ASTKGPSVFP LAPSSKS 147 M142-H08 LCQDIQMTQSPS SLSAFVGDRV TITCRASQPI DNYLNWYHQK PGKAPKLLIY AASRLQSGVP  60SRLSGSGFGT DFTLTISSLQ PEDFGNYYCQ QSYTVPYTFG GGTKVEIR 108 M142-H08 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS AYSMIWVRQA PGKGLEWVSY IRPSGGRTTY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARGG LLLWFRELKS NYFDYWGQGT 120LVTVSSASTK GPSVFPLAPS SKS 143 M145-D01 LCQDIQMTQSPA TLSLSPGERA TLSCRASQSV SSYLAWYQQK PGQAPRLLIY DASNRATGIP  60ARFSGSGSGT DFTLTISSLE PEDFAVYYCQ QRSNWPRGFT FGPGTKVDIK 110 M145-D01 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS YYHMSWVRQA PGKGLEWVSV ISPSGGSTKY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARGG SSDYAWGSYR RPYYFDYWGQ 120GTLVTVSSAS TKGPSVFPLA PSSKS 145 M145-D11 LCQSVLTQPPSV SVSPGQTASI TCSGDKLGDK YTSWYQQRPG QSPVLVIYQD IKRPSGIPER  60FSGSNSGNTA TLTISGTQAM DEADYYCQAW DSPNARVFGS GTKVTVL 107 M145-D11 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS HYRMSWVRQA PGKGLEWVSS IYPSGGRTVY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAKDK FEWRLLFRGI GNDAFDIWGQ 120GTMVTVSSAS TKGPSVFPLA PSSKS 145 M146-E12 LCQDIQMTQSPS SLSASVGDRV TITCRASGDI GNALGWYQQK PGKAPRLLIS DASTLQSGVP  60LRFSGSGSGT EFTLTISSLQ PEDFATYYCL QGYNYPRTFG QGTKLEIR 108 M146-E12 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS RYIMHWVRQA PGKGLEWVSS ISPSGGLTSY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAREF ENAYHYYYYG MDVWGQGTTV 120TVSSASTKGP SVFPLAPSSK S 141 M152-A12 LCQDIQMTQSPS SLSASVGDRV TITCRASQSI SSYLSWYQQR PGKAPNLLIY AASSLQSGVP  60SRFSGSGSGT DFTLTISSLQ PEDFATYYCQ QSISIPRTFG QGTKVEVK 108 M152-A12 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS PYFMGWVRQA PGKGLEWVSG IGPSGGSTTY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAREG PPYSSGWYRG LRQYHFDYWG 120QGTLVTVSSA STKGPSVFPL APSSKS 146 M160-G12 LCQDIQMTQSPS FLSASVGDRV TITCRASQGI SSYLAWYQQK PGKAPKLLIY AASTLQSGVP  60SRFSGSGSGT EFTLTISSLQ PEDFATYYCQ QLNSYPLTFG GGTKVEIK 108 M160-G12 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS HYLMTWVRQA PGKGLEWVSY ISPSGGHTIY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARVA RGIAARSRTS YFDYWGQGTL 120VTVSSASTKG PSVFPLAPSS KS 142 M161-C11 LCQSALTQPPSV SVSPGQTASI TCSGDKLGDK YVSWYQQRPG QSPVLVIYQD TKRPSGIPER  60FSGSNSGNTA TLTISGTQAV DEADYYCQAW DSSTYVFGGG TKVTVL 106 M161-C11 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS DYAMKWVRQA PGKGLEWVSS ISSSGGVTQY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAREE DYSSSWYSRR FDYYYGMDVW 120GQGTTVTVSS ASTKGPSVFP LAPSSKS 147 M162-A04 LCQDIQMTQSPS TLSASVGDRV TITCRASQSI SSWLAWYQQK PGKAPNLLIY KASTLESGVP  60SRFSGSGSGT EFTLTISSLQ PDDFATYYCQ QYNTYWTFGQ GTKVEIK 107 M162-A04 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS HYIMMWVRQA PGKGLEWVSG IYSSGGITVY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAYRR TGIPRRDAFD IWGQGTMVTV 120SSASTKGPSV FPLAPSSKS 139 X67-B03 LCQDIQMTQSPS SLSAFVGDRV TITCRASQPI DNYLNWYHQK PGKAPKLLIY AASRLQSGVP  60SRLSGSGFGT DFTLTISSLQ PEDFGNYYCQ QSYTVPYTFG GGTKVEIR 108 X67-B03 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS AYSMIWVRQA PGKGLEWVSY IRPSGGRTTY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARGG LLLWSRELKS NYFDYWGQGT 120LVTVSSASTK GPSVFPLAPS SKS 143 X67-C03 LCQDIQMTQSPS SLSAFVGDRV TITCRASQPI DNYLNWYHQK PGKAPKLLIY AASRLQSGVP  60SRLSGSGFGT DFTLTISSLQ PEDFGNYYCQ QSYTVPYTFG GGTKVEIR 108 X67-C03 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS AYSMIWVRQA PGKGLEWVSY IRPSGGRTTY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARGG LLLWMRELKS NYFDYWGQGT 120LVTVSSASTK GPSVFPLAPS SKS 143 X67-C09 LCQDIQMTQSPS SLSAFVGDRV TITCRASQPI DNYLNWYHQK PGKAPKLLIY AASRLQSGVP  60SRLSGSGFGT DFTLTISSLQ PEDFGNYYCQ QSYTVPYTFG GGTKVEIR 108 X67-C09 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS AYSMIWVRQA PGKGLEWVSY IRPSGGRTTY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARGG LLLWGRELKS NYFDYWGQGT 120LVTVSSASTK GPSVFPLAPS SKS 143 X67-D03 LCQDIQMTQSPS SLSAFVGDRV TITCRASQPI DNYLNWYHQK PGKAPKLLIY AASRLQSGVP  60SRLSGSGFGT DFTLTISSLQ PEDFGNYYCQ QSYTVPYTFG GGTKVEIR 108 X67-D03 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS AYSMIWVRQA PGKGLEWVSY IRPSGGRTTY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARGG LLLWNRELKS NYFDYWGQGT 120LVTVSSASTK GPSVFPLAPS SKS 143 X67-E04 LCQDIQMTQSPS SLSAFVGDRV TITCRASQPI DNYLNWYHQK PGKAPKLLIY AASRLQSGVP  60SRLSGSGFGT DFTLTISSLQ PEDFGNYYCQ QSYTVPYTFG GGTKVEIR 108 X67-E04 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS AYSMIWVRQA PGKGLEWVSY IRPSGGRTTY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARGG LLLWDRELKS NYFDYWGQGT 120LVTVSSASTK GPSVFPLAPS SKS 143 X67-F01 LCQDIQMTQSPS SLSAFVGDRV TITCRASQPI DNYLNWYHQK PGKAPKLLIY AASRLQSGVP  60SRLSGSGFGT DFTLTISSLQ PEDFGNYYCQ QSYTVPYTFG GGTKVEIR 108 X67-F01 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS AYSMIWVRQA PGKGLEWVSY IRPSGGRTTY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARGG LLLWQRELKS NYFDYWGQGT 120LVTVSSASTK GPSVFPLAPS SKS 143 X67-F10 LCQDIQMTQSPS SLSAFVGDRV TITCRASQPI DNYLNWYHQK PGKAPKLLIY AASRLQSGVP  60SRLSGSGFGT DFTLTISSLQ PEDFGNYYCQ QSYTVPYTFG GGTKVEIR 108 X67-F10 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS AYSMIWVRQA PGKGLEWVSY IRPSGGRTTY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARGG LLLWTRELKS NYFDYWGQGT 120LVTVSSASTK GPSVFPLAPS SKS 143 X67-G04 LCQDIQMTQSPS SLSAFVGDRV TITCRASQPI DNYLNWYHQK PGKAPKLLIY AASRLQSGVP  60SRLSGSGFGT DFTLTISSLQ PEDFGNYYCQ QSYTVPYTFG GGTKVEIR 108 X67-G04 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS AYSMIWVRQA PGKGLEWVSY IRPSGGRTTY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARGG LLLWARELKS NYFDYWGQGT 120LVTVSSASTK GPSVFPLAPS SKS 143 X67-H04 LCQDIQMTQSPS SLSAFVGDRV TITCRASQPI DNYLNWYHQK PGKAPKLLIY AASRLQSGVP  60SRLSGSGFGT DFTLTISSLQ PEDFGNYYCQ QSYTVPYTFG GGTKVEIR 108 X67-H04 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS AYSMIWVRQA PGKGLEWVSY IRPSGGRTTY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARGG LLLWERELKS NYFDYWGQGT 120LVTVSSASTK GPSVFPLAPS SKS 143Note: X81-B01 is a germilined IgG derived from X63-G06 which isshown in Table 7.

TABLE 2CDR Amino Acid Sequences and ELISA Signal of Antibody Binders of PKal(SEQ ID NOs: 294-743) HV- CDR1 LV-CDR2 (SEQ Human LV-CDR1 (SEQ IDLV-CDR3 ID HV-CDR3 pKal (SEQ ID NOs: (SEQ ID NOs: HV-CDR2 (SEQ IDInitial ELISA NOs: 369- NOs: 519- (SEQ ID NOs: NOs: Name (T/B) 294-368)443) 444-518) 593) 594-668) 669-743) M6-A06 11.7 RASQSISMYLN GTSSLQSQQSYSAPWT LYQMT GIWPSGGFTDYADSVKG VSTAVADNDY M6-A08 23.4 RASQRISFYLNGASSLQS QQTFSTPNT PYPMQ SISSSGGMTEYADSVKG DDYGGKGGAFDI M6-D03 15.5RASQSISSYLN AASSLQS QQSYSTLWT KYFMG VIGSSGGWTSYADSVKG VSTAVADNDY M6-D0816 RASQSISSYLN GASSLQS QQSYTRWT RYHMV SISPSGGWTNYADSVKG EMATIAGQFDPM6-G05 18.5 RASQSISTYLN NAFSMER QQSYTTPTT RYRMV SIYPSGGMTAYADSVKGDAVGIGDAFDI M8-C04 44.7 SGDKLGDKYTS QDSKRPS QAWDSSTV YYPMQYIYPSGGLTSYADSVKG LFYGSGSVGFEY M8-D05 11.9 RASQDISSWLV DASNLQS QQADGFPLTLYNMN SISPSGGFTDYADSVKG DLDLGILDY M8-E06 8.8 RASQSISSYLN AASSLQSQQSYSTLMYT HYFMT SIVPSGGMTQYADSVKG DSYSSSWFDI M8-G09 28.2 RASQGVSYYLAGASSLQS QQYNTYPPT LYEML VIYPSGGYTDYADSVKG SFSGFGEIDY M8-H04 3.3RASQYISTYLN GTSSLQS QQSFTTPFT GYWMG SISSSGGWTQYADSVKG DDEIAAGGAFDIM9-A03 14.4 RASQNIDIYLN GAYNLQS QQSYGTPV GYFMM SIYSSGGYTDYADSVKGEVAGTYAFDI M9-A08 5.5 RASQRISTYLN GASSLQS QQSYNTPRT AYEMWYIGSSGGSTSYADSVKG GNSSSFDAFDI M9-C08 10.9 RASQSISIYVN AASSLQR QQSFSTPLTHYGMV YIVPSGGLTYYADSVKG VDYTGDGLGY M9-C10 7.8 RASQGISSYLN GASSLQSQESYSTLFT LYPMQ SIGSSGGMTFYADSVKG EVGAAGFAFDI M9-D08 35.9 RASRTISFYLNGGSSLHS QQSFSSPWT WYKMM SIYPSGGWTNYADSVKG GSPWGDDAFDI M9-E04 18.8RASQSISGYLN AASNLQT QQSHTPPKT EYDMM SIGSSGGMTYYADSVKG DQVAAAAIDY M9-F0810.9 RASQSISSYLN AASSLQS QQSYSTPPYT PYAMT VIYPSGGFTDYADSVKG ASGSYLDAFDIM9-F09 7 RASQSISSYLN AASSLQS QQTYTTPWT SYPMG RISSSGGMTIYADSVKG DDWNVGMDVM9-F10 8.4 RASQSINTYLN AASTLES QQSYSTPYT DYDME SISPSGGSTIYADSVKGQGLLTAFDI M9-G08 4.8 RASQSISSYLN AASSLQS QQSYSTPIT YYTMLSIYPSGGFTMYADSVKG VDTAMAMIDY M9-H02 3.5 RASRSIATYLN GASTLQS QQSFSDPYTAYMMI VIYPSGGVTMYADSVKG GTVGASDAFDI M9-H03 4.4 SGDKLGNRYTS QDNKRPSQALDSNTYV WYSMG YIVPSGGYTMYADSVKG DPGVSYYYYGMDV M9-H04 16.1 RASQSISSYLNAASSLQS QQSYSTPPT AYTMW SIWPSGGSTFYADSVKG TYDSSAGEVDY M10-A03 33.7RASQRISFYLN GASSLQS QQTFSTPNT PYPMQ SISSSGGMTEYADSVKG DDYGGKGGAFDIM10-A12 20.8 RASRDISVYLN GASSLQS QQSYSIPFT LYLMH SIYSSGGFTTYADSVKGDTDYGMDV M10-B09 14.1 RASQSISTYLN GASSLQS QQSFSTPWT WYEMSRIWPSGGVTMYADSVKG TSITTVGMDV M10-C11 5.3 RASQSISIYLN AASTLQS QQSHSIPPTMYPMM YISPSGGMTDYADSVKG VAGSSDAFDI M10-D11 6.4 RSSQSLLHSNGYNYLD LGSNRASMQALQTPLT AYPMN RISSSGGNTSYADSVKG GYLGY M10-E06 32.8 RASQSISTYLN GASSLQSQQSYSDPYT LYRMF SIWSSGGPTMYADSVKG EYPSTYYFDY M10-F09 4.8 RASQTIDDDLIAASSLQS QQSYNIPRT NYDMM YISPSGGFTRYADSVKG DIYYYNWGPSHYFDS M10-G09 7.1RASQSISGYIN AASSLQS QQYVSYPFT QYGMQ SIRSSGGATRYADSVKG DGYYDSSGYPDYM11-A10 25 RASQSIDTYLN DASNL QHYLYAPYS NYWMM GIGSSGGFTSYADSVKGGSYSDYGVFES M11-E01 11.7 RASQSISSYLN AASSLQS QQSYSTPPT TYEMYGIGSSGGMTMYADSVKG EQPGIAALQF M11-E04 43.2 RASQSISIYLT GAATLQT QQTFSLPRTMYHMN GIVSSGGVTFYADSVKG ITTVTTGGAFDI M11-E05 41.4 RTSQTINNYLN ATHTLESQQSFAFPYT WYTMG WIYFGGLTTYADSVKG LGGPLDAFDI M11-E06 12.6 RASRGIGTYLNAASSLET QESFTNVYN QYAMH SIYPSGGFTLYADSVKG GGWLAGGELLN M11-G09 23.6RTSQGINHYLN AASELQT QQTYTSPYT LYNMT YIYPSGGGTHYADSVKG DTGFWSADAFDIM11-G12 4.9 RASQTISVYVN GASSLQS QQSYSIPFT QYPMN SISSSGGFTTYADSVKGEEQQGGFDY M12-A08 40.4 RASQSISRYLN AASTLET QQSYSTPYT WYYMGWIVSSGGLTLYADSVKG TTVTTGDAFDI M12-B04 18 RASQGIRNDLG AASILQS LQDYEYPLTLYSMY RIRPSGGGTVYADSVKG DPLYSSGDV M12-C09 7 RASQSIGIYLN GASSLQSQHSYSTPFT SYAMV SIGSSGGFTLYADSVKG MNLGGGDAFDI M12-C10 8.3 SGDKLGEKYVSQDNKRPS QAWDSYTVV DYEMH GISPSGGKTQYADSVKG DLKWGGRGSPDWYFDL M12-D10 9.9RASQSISSYLN AASSLQS QQSYSTPPT NYPMD SISSSGGWTNYADSVKG DTSGSYLGFDYM12-E06 48 RASQSISTYLN GAFSLQS QQSHSTPPT QYKML GIGPSGGLTAYADSVKGAPWFGELGMDV M27-A10 3.2 RASQSISAYLN YGVGSLQS QQGYTTPVT WYRMDSIWPSGGLTSYADSVKG GWAPGGDAFDI M27-B01 33.1 RASQSISSYLN AASSLQS QQSYSTPYTDYTMW SISSSGGITFYADSVKG SADTAMGGAFDI M27-B12 2.3 SGDKLGDEYAA QDRKRPSQAWGKRNVV WYQMM SISPSGGITEYADSVKG DRSSGWYYYGMDV M27-E03 35.9 RASQSISSYLNAASSLQS QQSYSTPRT SYMMH GIYPSGGWTDYADSVKG LVAGLDAFDI M27-F04 10.5RASQSISSYLN AASSLQS QQSYSTPPT WYPMT SIGPSGGQTIYADSVKG EYGDYGGGFDPM27-F11 10 RASQGISSYLA AASSLQS QQSYNTLRT SYHMM SIYPSGGATMYADSVKGDGYHYGDYTYFQH M27-G01 31.4 RASQSISTYLN GASSLQS QQSYSDPYT LYRMFSIWSSGGPTMYADSVKG EYPSTYYFDY M27-G04 4.1 RASQRISYYLT AASSLES QQAFSTPFTAYYMV YISPSGGQTQYADSVKG EAISSSSFDY M27-G09 2.2 RTRQSISNYLN AASSLQSQQSYDIPFT EYDMA YIVSSGGFTSYADSVKG WAGWIAAADY M27-H10 12.4 RASQSISNYLNAASSLQS QQSYSTPQT AYQMA VIYSSGGYTDYADSVKG HNWNDGAFDI M28-A01 19RASQSISSYLN AASSLQS QQSYSTLT WYAMH GIYSSGGYTKYADSVKG DLSNGDDVFDI M28-C032.2 RASQSINFYLN VASSLES LQSYSAPYT YYQMG SIYPSGGMTDYADSVKG GSPWGDDAFDIM28-D02 3.7 RTSRRIGTYLN GASSLQS QQSFSSPWT WYPMQ YIYPSGGGTDYADSVKGSSGWLGDAFDI M28-D12 41.6 RASQSIATYLN AASSLQS XSYSTRET WYTMHVIYPSGGPTSYADSVKG DGSGSYLGFDY M28-E01 41 RASQSISSYLN AASSLQS QQTYTTPWTSYPMG RISSSGGMTIYADSVKG DDWNVGMDV M28-E11 29.3 RASQDISNWLA AASSLQTQQSYSLPWT LYDMT GISSSGGVTIYADSVKG TYYYDSSGYADAFDI M28-F01 1.5RASQSINTYLN AASTLES QQSYSTPPT VYLMH GISPSGGYTQYADSVKG PGGLDAFDI M28-F0531.4 RASQSISSYLN AASSLQS XSYSTPLT RYIMW GIYSSGGYTQYADSVKG ELEGLGGFDYM28-F07 33 RASQGISSWLA ATSGLQS QQAKSFPLT DYTMY SIVPSGGHTLYADSVKGDHLSSWYGGFFDY M29-C07 5.2 RASQSISSYLN AASSLQS XSYSTRYT GYDMMVISSSGGNTAYADSVKG ESSGLYYFDY M29-D10 23.6 RASQSITIYLN GASNLHS QQSYDTPLTWYPMY SIGSSGGPTPYADSVKG WADYGGSLDY M29-E02 2 SGSSSNIGNNAVS YDDLLPSAAWDDSLNGFV RYPMM VIYPSGGDTFYADSVKG GDDYLWEAAVY M29-G08 40.4 RASQNIGNDVAHASTRAY QQFYDWPAHT YYHMW GISPSGGFTFYADSVKG DYYYDSSGYSPLGY M29-G10 16.4RASQSISIYLN GASQLES QQSYNVPYT FYKMI SISSSGGSTQYADSVKG DRVDLGYLDY M74-A078.6 RTSQNINTYLN GVSSLHR QQSYSSPWT QYLMM SIYPSGGYTSYADSVKG VSTAVADNDYM76-F02 6.4 RASQTIDNYLH DASSLQS QQSYDTPQYT LYDMN GISPSGGQTMYADSVKGQPMISAFDI M76-G02 10.3 RASQSISSYLN AASSLQS QQSYSTPPWT LYAMWYISSSGGFTSYADSVKG YRVGVAATDY M76-G06 11.8 RASQSISTYLN AASSLQS XSYSTPHTGYIMH WIYPSGGWTEYADSVKG DAPGVGAIDY M76-H02 13.4 RASQDISVYLN GGASLQSQQSYSLPFT MYWMQ YIYPSGGPTKYADSVKG PSGSYGDAFDI M77-C07 16.1 RASQNISSYLNAASSLQS XSYSTPRT LYING GIYPSGGFTMYADSVKG ESSGVAAPDY M77-H04 7.6RSSQSLLHSRGYNYLD LGSNRAS MQALQRRT YYTMI GIRSSGGGTRYADSVKGDGSRYSYGSIYYYYGMDA Abbreviations used: “T/B” is the ELISA signalobtained using of the “target” (biotinylated plasma kallikrein) dividedby the ELISA signal of the “background” (streptavidin); both of whichwere coated on microtiter plates. “nd” is not determined. The symbol “q”refers to the amber suppressible stop codon (TAG), which is translatedas glutamine (Q) in strains of E. coli such as the TG1 cells that wereused to express the Fab fragments.

Amino acid sequences of light chain (LC) and heavy chain (HC) variabledomain of pKal antibody binders are shown below. (SEQ ID NOs:744-893numbered in order from top to bottom)

M6-A06 LCQDIQMTQSPS SLSASVGDSV TISCRASQSI SMYLNWYQHK PGKAPKLLIY GTSSLQSGVP  60SRFSGSGPGG TDFTLTISSL QPEDFATYYC QQSYSAPWTF GQGTKVEIK 109 M6-A06 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS LYQMTWVRQA PGKGLEWVSG IWPSGGFTDY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARVS TAVADNDYWG QGTLVTVSSA 120STKGPSVFPL APSSKS 136 M6-A08 LCQDIQMTQSPS SLSASVGDRV TITCRASQRI SFYLNWFQQK PGKAPNLLIY GASSLQSGVP  60SRFSGSGSGT DFTLTISSLQ PKDFGTYYCQ QTFSTPNTFG QGTKLEIK 108 M6-A08 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS PYPMQWVRQA PGKGLEWVSS ISSSGGMTEY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARDD YGGKGGAFDI WGQGTMVTVS 120SASTKGPSVF PLAPSSKS 138 M6-D03 LCQDIQMTQSPS SLSASVGDRV TITCRASQSI SSYLNWYQQK PGKAPKLLIY AASSLQSGVP  60SRFSGSGSGT DFTLTISSLQ PEDFATYYCQ QSYSTLWTFG QGTKVEIK 108 M6-D03 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS KYFMGWVRQA PGKGLEWVSV IGSSGGWTSY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARVS TAVADNDYWG QGTLVTVSSA 120STKGPSVFPL APSSKS 136 M6-D08 LCQDIQMTQSPS SLSASVGDRV TITCRASQSI SSYLNWYqQK PGKAPKLLIY GASSLQSGVP  60SRFSGSGSGT DFTLTISSLQ PEDSATYYCQ QSYTRWTFGQ GTKVEIK 107 M6-D08 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS RYHMVWVRQA PGKGLEWVSS ISPSGGWTNY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAMYYCAREM ATIAGQFDPW GQGTLVTVSS 120ASTKGPSVFP LAPSSKS 137 M6-G05 LCQDIQMTQSPS SLSASVGDRV TITCRASQSI STYLNWYQLK PGKAPKLLIY NAFSMERGVP  60STISGSGSGT DFTLTISSLQ PEDFATYYCQ QSYTTPTTFG QGTKVEIK 108 M6-G05 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS RYRMVWVRQA PGKGLEWVSS IYPSGGMTAY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARDA VGIGDAFDIW GQGTMVTVSS 120ASTKGPSVFP LAPSSKS 137 M8-C04 LCQSALTQPPSV SVSPGQTASI TCSGDKLGDK YTSWHQQKPG QSPVLVIYQD SKRPSGIPER  60FSGSNSGNTA TLTISGTQAM DEADYYCQAW DSSTVFGGGT RLTVL 105 M8-C04 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS YYPMQWVRQA PGKGLEWVSY IYPSGGLTSY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARLF YGSGSVGFEY WGQGTLVTVS 120SASTKGPSVF PLAPSSKS 138 M8-D05 LCQDIQMTQSPS FVSASVGDRV TITCRASQDI SSWLVWYQQK PGKGPKLLIY DASNLQSGVP  60SRFSGGGSGT HFTLTISSLQ PEDFATYYCQ QADGFPLTFG GGTKVEMK 108 M8-D05 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS LYNMNWVRQA PGKGLEWVSS ISPSGGFTDY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARDL DLGILDYWGQ GTLVTVSSAS 120TKGPSVFPLA PSSKS 135 M8-E06 LCQDIQMTQSPS SLSASVGDRV TITCRASQSI SSYLNWYQQK PGKAPKLLIY AASSLQSGVP  60SRFSGSGSGT DFTLTISSLQ PEDFATYYCQ QSYSTLMYTF GQGTKLEIK 109 M8-E06 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS HYFMTWVRQA PGKGLEWVSS IVPSGGMTQY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARDS YSSSWFDIWG QGTMVTVSSA 120STKGPSVFPL APSSKS 136 M8-G09 LCQDIQMTQSPS SLSASVGDTV TITCRASQGV SYYLAWFQQK PGKAPKSLIY GASSLQSGVP  60SKFSGSGSGT VFTLTISSLQ PDDFATYYCQ QYNTYPPTFG QGTRLDIK 108 M8-G09 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS LYEMLWVRQA PGKGLEWVSV IYPSGGYTDY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED MAVYYCARSF SGFGEIDYWG QGTLVTVSSA 120STKGPSVFPL APSSKS 136 M8-H04 LCQDIQMTQSPS SLSASIGDRV TITCRASQYI STYLNWYEQK PGKAPKLLIY GTSSLQSGVP  60SRFSGSGSGT EFSLTISSLQ PEDFATYYCQ QSFTTPFTFG QGTKLEIK 108 M8-H04 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS GYWMGWVRQA PGKGLEWVSS ISSSGGWTQY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TATYYCARDD EIAAGGAFDI WGQGAMVTVS 120SASTKGPSVF PLAPSSKS 138 M9-A03 LCQDIQMTQSPS SLSASLGDRV TITCRASQNI DIYLNWYQQT PGKAPKLLIY GAYNLQSGVP  60SRFSGSGSGT DFTLTISSLQ PEDFGTYYCQ QSYGTPVFGQ GTKLEIK 107 M9-A03 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS GYFMMWVRQA PGKGLEWVSS IYSSGGYTDY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAREV AGTYAFDIWG QGTMVTVSSA 120STKGPSVFPL APSSKS 136 M9-A08 LCQDIQMTQSPS SLSASVGDRV TVTCRASQRI STYLNWYQQK PGKAPKLLIS GASSLQSGVP  60SRFSGSGSGT DFTLTISSLQ PDDFATYYCQ QSYNTPRTFG QGTKVEIR 108 M9-A08 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS AYEMWWVRQA PGKGLEWVSY IGSSGGSTSY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCTGGN SSSFDAFDIW GQGTMVTVSS 120ASTKGPSVFP LAPSSKS 137 M9-C08 LCQDIQMTQSPS SLSASVGDRV TITCRASQSI SIYVNWYQQK PGKAPNLLIF AASSLQRGVP  60SRFSGSGSGA DFTLTISSLQ PEDFATYYCQ QSFSTPLTFG GGTKVEIK 108 M9-C08 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS HYGMVWVRQA PGKGLEWVSY IVPSGGLTYY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARVD YTGDGLGYWG QGTLVTVSSA 120STKGPSVFPL APSSKS 136 M9-C10 LCQDIQMTQSPS SLSASVGDRV TITCRASQGI SSYLNWYQQK PGNAPNLLIY GASSLQSGVP  60SRFSGSGSGT DFTLTISSLQ PEDFATYYCQ ESYSTLFTFG PGTTVEIK 108 M9-C10 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS LYPMQWVRQA PGKGLEWVSS IGSSGGMTFY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCTREV GAAGFAFDIW GQGTMVTVSS 120ASTKGPSVFP LAPSSKS 137 M9-D08 LCQDIQMTQSPS SLSASVGDRV TLTCRASRTI SFYLNWYQQK AGKAPELLIY GGSSLHSGVP  60SRFSGSGSGT DFSLTISNLQ PEDIAVYYCQ QSFSSPWTFG QGTKVEIK 108 M9-D08 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS WYKMMWVRQA PGKGLEWVSS IYPSGGWTNY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCTRGS PWGDDAFDIW GQGTMVTVSS 120ASTKGPSVFP LAPSSKS 137 M9-E04 LCQDIQMIQSPS SLSASVGDRV TITCRASQSI SGYLNWYQQR SGKAPKLLIF AASNLQTGVP  60SRFSGSGSGT DFTLTINNLQ PEDFATYYCQ QSHTPPKTFG PGTKVDIK 108 M9-E04 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS EYDMMWVRQA PGKGLEWVSS IGSSGGMTYY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARDQ VAAAAIDYWG QGTLVTVSSA 120STKGPSVFPL APSSKS 136 M9-F08 LCQDIQMTqSPS SLSASVGDRV TITCRASQSI SSYLNWYQQK PGKAPKLLIY AASSLQSGVP  60SRFSGSGSGT DFTLTISSLQ PEDFATYYCQ QSYSTPPYTF GQGTKLEIK 109 M9-F08 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS PYAMTWVRQA PGKGLEWVSV IYPSGGFTDY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAMYYCARAS GSYLDAFDIW GQGTMVTVSS 120ASTKGPSVFP LAPSSKS 137 M9-F09 LCQDIQMTQSPS SLSASVGDRV TITCRASQSI SSYLNWYQQK PGKAPKLLIY AASSLQSGVP  60SKFSGSGSGT DYTLTISSLQ PEDFATYYCQ QTYTTPWTFG QGTKVEIK 108 M9-F09 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS SYPMGWVRQA PGKGLEWVSR ISSSGGMTIY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARDD WNVGMDVWGQ GTTVTVSSAS 120TKGPSVFPLA PSSKS 135 M9-F10 LCQDIQMTQSPS SLSASVGDRV TITCRASQSI NTYLNWYQQK PGKAPKVLIH AASTLESGVP  60SRFSGSGSGT DFTLTISSLQ PEDFATYYCQ QSYSTPYTFG QGTKLEVR 108 M9-F10 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS DYDMEWVRQA PGKGLEWVSS ISPSGGSTIY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARQG LLTAFDIWGQ GTMVTVSSAS 120TKGPSVFPLA PSSKS 135 M9-G08 LCQDIQMTQSPS SLSASVGDRV TITCRASQSI SSYLNWYQQK PGKAPKLLIY AASSLQSGVP  60SRFSGSGSGT DFTLTISSLQ PEDFATYYCQ QSYSTPITFG GGTKVEIK 108 M9-G08 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS YYTMLWVRQA PGKGLEWVSS IYPSGGFTMY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARVD TAMAMIDYWG QGTLVTVSSA 120STKGPSVFPL APSSKS 136 M9-H02 LCQDIQMTQSPS SLSASVGDRV IITCRASRSI ATYLNWYQQK PGKAPNLLIF GASTLQSGVP  60SRFSGSGSGT DFTLTISDLQ PEDFATYYCQ QSFSDPYTFG QGTNLEMK 108 M9-H02 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS AYMMIWVRQA PGKGLEWVSV IYPSGGVTMY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARGT VGASDAFDIW GQGTMVTVSS 120ASTKGPSVFP LAPSSKS 137 M9-H03 LCQYELTQAPSV SVAPGQTASI TCSGDKLGNR YTSWYQQKPG QSPVLVIFQD NKRPSGIPER  60FSGSNSGNTA TLTISGTQAM DEADYYCQAL DSNTYVFGTG TKVTVL 106 M9-H03 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS WYSMGWVRQA PGKGLEWVSY IVPSGGYTMY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARDP GVSYYYYGMD VWGQGTTVTV 120SSASTKGPSV FPLAPSSKS 139 M9-H04 LCQDIQMTQSPS SLSASVGDRV TITCRASQSI SSYLNWYQQK PGKAPKLLIY AASSLQSGVP  60SRFSGSGSGT DFTLTISSLQ PEDFATYYCQ QSYSTPPTFG QGTRLEIK 108 M9-H04 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS AYTMWWVRQA PGKGLEWVSS IWPSGGSTFY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARTY DSSAGEVDYW GQGTLVTVSS 120ASTKGPSVFP LAPSSKS 137 M10-A03 LCQDIQMTQSPS SLSASVGDRV TITCRASQRI SFYLNWFQQK PGKAPNLLIY GASSLQSGVP  60SRFSGSGSGT DFTLTISSLQ PKDFGTYYCQ QTFSTPNTFG QGTKLEIK 108 M10-A03 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS PYPMQWVRQA PGKGLEWVSS ISSSGGMTEY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARDD YGGKGGAFDI WGQGTMVTVS 120SASTKGPSVF PLAPSSKS 138 M10-A12 LCQDIQMTQSPL SLSAFVGDRV TITCRASRDI SVYLNWYQLK SGKAPKLLIY GASSLQSGVP  60SRFSGSGSGT DFTLTITSLQ PEDFATYYCQ QSYSIPFTFG GGTKVETK 108 M10-A12 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS LYLMHWVRQA PGKGLEWVSS IYSSGGFTTY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARDT DYGMDVWGQG TTVTVSSAST 120KGPSVFPLAP SSKS 134 M10-B09 LCQDIQMTQSPS SLSASVGDGV TITCRASQSI STYLNWYQQR PGKAPKLLIY GASSLQSGVP  60SRFSGSGSGT DFTLTISSLQ REDFATYYCQ QSFSTPWTFG QGTRVEIK 108 M10-B09 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS WYEMSWVRQA PGKGLEWVSR IWPSGGVTMY  60ADSVKGRFTI SRDNSKNTLY LqMNSLRAED TAVYYCTRTS ITTVGMDVWG QGTTVTVSSA 120STKGPSVFPL APSSKS 136 M10-C11 LCQDIQMTQSPS SLSASVGDRV TITCRASQSI SIYLNWYQQK PEKAPKLLIF AASTLQSGVP  60SRFSGSGSGT DFTLTISNLQ PEDFATYYCQ QSHSIPPTFG LGTKVEVK 108 M10-C11 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS MYPMMWVRQA PGKGLEWVSY ISPSGGMTDY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED MAVYYCARVA GSSDAFDIWG QGTMVTVSSA 120STKGPSVFPL APSSKS 136 M10-D11 LCQDIQMTQSPL SLPVTPGEPA SISCRSSQSL LHSNGYNYLD WYLQKPGQSP QLLIYLGSNR  60ASGVPDRFSG SGSGTDFTLK ISRVEAEDVG VYYCMQALQT PLTFGPGTKV HIK 113M10-D11 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS AYPMNWVRQA PGKGLEWVSR ISSSGGNTSY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCALGY LGYWGQGTLV TVSSASTKGP 120SVFPLAPSSK S 131 M10-E06 LCQDIQMTQSPS SLSASVGDRV TITCRASQSI STYLNWYQQK PGKAPKLLIY GASSLQSGVP  60SRFSGSGSGT DFTLTISSLQ PEDFTIYYCQ QSYSDPYTFG QGTKLDIK 108 M10-E06 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS LYRMFWVRQA PGKGLEWVSS IWSSGGPTMY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAREY PSTYYFDYWG QGTLVTVSSA 120STKGPSVFPL APSSKS 136 M10-F09 LCQDIQMTQSPS SLSASVGDRV TITCRASQTI DDDLIWYQQK PGRAPKLLIY AASSLQSGVP  60SRFSGSGSGT DFTLTITSLQ PEDFATYYCQ QSYNIPRTFG QGTKLESK 108 M10-F09 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS NYDMMWVRQA PGKGLEWVSY ISPSGGFTRY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TATYYCAKDI YYYNWGPSHY FDSWGQGTLV 120TVSSASTKGP SVFPLAPSSK S 141 M10-G09 LCQDIQMTQSPS SLSASVGDSV TITCRASQSI SGYINWYQQK AGKAPKLLIY AASSLQSGVP  60SRFSGSGSGT HFTLTISSLQ PEDFATYYCQ QYVSYPFTFG PGTKVDIK 108 M10-G09 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS QYGMQWVRQA PGKGLEWVSS IRSSGGATRY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARDG YYDSSGYPDY WGQGTLVTVS 120SASTKGPSVF PLAPSSKS 138 M11-A10 LCQDIQMTQSPS SLSASVGDRV AITCRASQSI DTYLNWYQQK PGKAPKLLIY DASNLEIGVP  60SRFSGSGSGT DFTFIINSLQ PEDVATYYCQ HYLYAPYSFG QGTKLEIK 108 M11-A10 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS NYWMMWVRQA PGKGLEWVSG IGSSGGFTSY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAKGS YSDYGVFESW GQGTLVTVSS 120ASTKGPSVFP LAPSSKS 137 M11-E01 LCQDIQMTQSPS SLSASVGDRV TITCRASQSI SSYLNWYQQK PGKAPKLLIY AASSLQSGVP  60SRFSGSGSGT DFTLTISSLQ PEDFATYYCQ QSYSTPPTFG QGTKVEIK 108 M11-E01 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS TYEMYWVRQA PGKGLEWVSG IGSSGGMTMY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAMYYCAREQ PGIAALQFWG QGTLVTVSSA 120STKGPSVFPL APSSKS 136 M11-E04 LCQDIQMTQSPS SLSASVGDRV TITCRASQSI SIYLTWYQHR PGKAPNLLIY GAATLQTGVP  60SRFSGSGSGT DFTLTIRGLQ PEDFATYYCQ QTFSLPRTFG QGTKLEIK 108 M11-E04 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS MYHMNWVRQA PGKGLEWVSG IVSSGGVTFY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARIT TVTTGGAFDI WGQGTMVTVS 120SASTKGPSVF PLAPSSKS 138 M11-E05 LCQDIQMTQSPS SLSASVGDTV TITCRTSQTI NNYLNWYQQR PGEAPKVLIY ATHTLESGVP  60SRFSGSGSGT DFTLTIGSLQ PEDFATYYCQ QSFAFPYTFG QGTKVEIT 108 M11-E05 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS WYTMGWVRQA PGKGLEWVSW IYFGGLTTYA  60DSVKGRFTIS RDNSKNTLYL QMNSLRAEDT AVYYCARLGG PLDAFDIWGQ GTMVTVSSAS 120TKGPSVFPLA PSSKS 135 M11-E06 LCQDIQMTQSPS SLSASIGDRV TISCRASRGI GTYLNWYQQH AGKAPKLLIR AASSLETGVP  60PRFSGSGSGT DFTLTISSLQ SDDFATYYCQ ESFTNVYNFG QGTKLEIK 108 M11-E06 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS QYAMHWVRQA PGKGLEWVSS IYPSGGFTLY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAMYYCARGG WLAGGELLNW GQGTLVTVSS 120ASTKGPSVFP LAPSSKS 137 M11-G09 LCQDIQMTQSPS SLSASVGDRV TITCRTSQGI NHYLNWYQQK PGKAPKILVF AASELQTGVP  60SRFSGTGSGT SYTLTITSLQ PEDVATYYCQ QTYTSPYTFG QGTKLEVK 108 M11-G09 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS LYNMTWVRQA PGKGLEWVSY IYPSGGGTHY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARDT GFWSADAFDI WGQGTMVTVS 120SASTKGPSVF PLAPSSKS 138 M11-G12 LCQDIQMTQSPS SLSAFVGDRV SITCRASQTI SVYVNWYQHK SGQAPKLLIY GASSLQSGVP  60SRFSGSGSGT DFTLTISSLQ PEDFATYFCQ QSYSIPFTFG GGTDVQIR 108 M11-G12 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS QYPMNWVRQA PGKGLEWVSS ISSSGGFTTY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAREE QQGGFDYWGQ GTLVTVSSAS 120TKGPSVFPLA PSSKS 135 M12-A08 LCQDIQMTQSPS SLSASVGDRV TITCRASQSI SRYLNWYQQK PGKAPKLLIY AASTLETGVP  60SRFSGSGSGT DFTLTITTLQ PEDFVIYYCQ QSYSTPYTFG QGTKLEIK 108 M12-A08 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS WYYMGWVRQA PGKGLEWVSW IVSSGGLTLY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAMYYCARTT VTTGDAFDIW GQGTMVTVSS 120ASTKGPSVFP LAPSSKS 137 M12-B04 LCQDIQMTQSPS SLSASVGDRV TITCRASQGI RNDLGWYQHK PGKAPKLLIY AASILQSGVP  60SRFSGTASGT DFTLTISSLQ PEDFATYFCL QDYEYPLTFG GGTKLDIK 108 M12-B04 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS LYSMYWVRQA PGKGLEWVSR IRPSGGGTVY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARDP LYSSGDVWGQ GTTVTVSSAS 120TKGPSVFPLA PSSKS 135 M12-C09 LCQDIQMTQSPS SLSASVGDRV TITCRASQSI GIYLNWYHQK PGKAPNLLIY GASSLQSGVP  60SRFSGSGSGT DFTLTISSLQ PGDFATYYCQ HSYSTPFTFG GGTKVEIK 108 M12-C09 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS SYAMVWVRQA PGKGLEWVSS IGSSGGFTLY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCASMN LGGGDAFDIW GQGTMVTVSS 120ASTKGPSVFP LAPSSKS 137 M12-C10 LCQSALTQPPSV SVSPGQTASI TCSGDKLGEK YVSWYQQKPG QSPVVVIYQD NKRPSGIPER  60FSGSNSGNTA TLTISGTQAV DEADYYCQAW DSYTVVFGGG SKLTVLGQPK 110 M12-C10 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS DYEMHWVRQA PGKGLEWVSG ISPSGGKTQY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARDL KWGGRGSPDW YFDLWGRGTL 120VTVSSASTKG PSVFPLAPSS KS 142 M12-D10 LCQDIQMTQSPS SLSASVGDRV TITCRASQSI SSYLNWYQQK PGKAPKLLIY AASSLQSGVP  60SRFSGSGSGT DFTLTISSLQ PEDFATYYCQ QSYSTPPTFG GGTKVEIK 108 M12-D10 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS NYPMDWVRQA PGKGLEWVSS ISSSGGWTNY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCATDT SGSYLGFDYW GQGTLVTVSS 120ASTKGPSVFP LAPSSKS 137 M12-E06 LCQDIQMTQSPS SLSASVGDRV SITCRASQSI STYLNWYQHK PGKAPTLLIY GAFSLQSGVP  60SRFSGSGSGT DFALTISSLQ PEDFATYYCQ QSHSTPPTFG QGTRVEIK 108 M12-E06 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS QYKMLWVRQA PGKGLEWVSG IGPSGGLTAY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARAP WFGELGMDVW GQGTTVTVSS 120ASTKGPSVFP LAPSSKS 137 M27-A10 LCQDIQMTQSPS SLSASVGDRV TITCRASQSI SAYLNWYQQK PGKAPQLLMY GVGSLQSGVP  60SRFSGSGSGT DFTLTISSLQ PEDFATYFCQ QGYTTPVTFG GGTKVEIK 108 M27-A10 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS WYRMDWVRQA PGKGLEWVSS IWPSGGLTSY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARGW APGGDAFDIW GQGTMVTVSS 120ASTKGPSVFP LAPSSKS 137 M27-B01 LCQDIQMTQSPS SLSASVGDRV TITCRASQSI SSYLNWYQQK PGKAPKLLIY AASSLQSGVP  60SRFSGSGSGT DFTLTISSLQ PEDFATYYCQ QSYSTPYTFG QGTKLEIK 108 M27-B01 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS DYTMWWVRQA PGKGLEWVSS ISSSGGITFY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARSA DTAMGGAFDI WGQGTMVTVS 120SASTKGPSVF PLAPSSKS 138 M27-B12 LCQYELTQPPAV SVSPGQTATI TCSGDKLGDE YAAWYQQKPG QSPVLVIYQD RKRPSGIPER  60FSGSNFGNTA TLTITGTQVM DEADYYCQAW GKRNVVFGGG TKLTVL 106 M27-B12 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS WYQMMWVRQA PGKGLEWVSS ISPSGGITEY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARDR SSGWYYYGMD VWGQGTTVTV 120SSASTKGPSV FPLAPSSKS 139 M27-E03 LCQDIQMTQSPS SLSASVGDRV TITCRASQSI SSYLNWYQQK PGKAPKLLIY AASSLQSGVP  60SRFSGSGSGT DFTLTISSLQ PEDFATYYCQ QSYSTPRTFG QGTKVEIK 108 M27-E03 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS SYMMHWVRQA PGKGLEWVSG IYPSGGWTDY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TATYYCARLV AGLDAFDIWG QGTMVTVSSA 120STKGPSVFPL APSSKS 136 M27-F04 LCQDIQMTQSPS SLSASVGDRV TITCRASQSI SSYLNWYQQK PGKAPKLLIY AASSLQSGVP  60SRFSGSGSGT DFTLTISSLQ PEDFATYYCQ QSYSTPPTFG QGTKVEIK 108 M27-F04 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS WYPMTWVRQA PGKGLEWVSS IGPSGGQTIY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCTTEY GDYGGGFDPW GQGTLVTVSS 120ASTKGPSVFP LAPSSKS 137 M27-F11 LCQDIQMTQSPS FLSASVGDRV TITCRASQGI SSYLAWYQQK PGKAPKLLIY AASSLQSGVP  60SRFSGSGSGT DFTLTISSLQ PEDFATYYCQ QSYNTLRTFG PGTKVDLK 108 M27-F11 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS SYHMMWVRQA PGKGLEWVSS IYPSGGATMY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAMYYCARDG YHYGDYTYFQ HWGQGTLVTV 120SSASTKGPSV FPLAPSSKS 139 M27-G01 LCQDIQMTQSPS SLSASVGDRV TITCRASQSI STYLNWYQQK PGKAPKLLIY GASSLQSGVP  60SRFSGSGSGT DFTLTISSLQ PEDFTIYYCQ QSYSDPYTFG QGTKLDIK 108 M27-G01 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS LYRMFWVRQA PGKGLEWVSS IWSSGGPTMY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAREY PSTYYFDYWG QGTLVTVSSA 120STKGPSVFPL APSSKS 136 M27-G04 LCQDIQMTQSPS SLSASVGDRV TITCRASQRI SYYLTWYQQK PGKVPKLLIY AASSLESGVP  60SRFSGSGSGT DFTLTISNLQ PEDFATYYCQ QAFSTPFTFG GGTKVEIK 108 M27-G04 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS AYYMVWVRQA PGKGLEWVSY ISPSGGQTQY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAREA ISSSSFDYWG QGTLVTVSSA 120STKGPSVFPL APSSKS 136 M27-G09 LCQDIQMTQSPS SVSASVGDRI TITCRTRQSI SNYLNWYQQK PGEPPKLLIF AASSLQSGVP  60SRFSGSGTGT EFTLTISSLQ PEDLAIYYCQ QSYDIPFTFG QGTKLEIK 108 M27-G09 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS EYDMAWVRQA PGKGLEWVSY IVSSGGFTSY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCTTWA GWIAAADYWG QGTLVTVSSA 120STKGPSVFPL APSSKS 136 M27-H10 LCQDIQMTQSPS SLSASVGDRV TITCRASQSI SNYLNWYQQK PGKAPKFLIY AASSLQSGVP  60SRFSGSGSGT DFTLSISSLQ PEDFATYYCQ QSYSTPQTFG QGTKVEMK 108 M27-H10 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS AYQMAWVRQA PGKGLEWVSV IYSSGGYTDY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARHN WNDGAFDIWG QGTMVTVSSA 120STKGPSVFPL APSSKS 136 M28-A01 LCQDIQMTQSPS SLSASVGDRV TITCRASQSI SSYLNWYQQK PGKAPKLLIY AASSLQSGVP  60SRFSGSGSGT DFTLTISSLQ PEDFATYYCQ QSYSTLTFGG GTKVEIK 107 M28-A01 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS WYAMHWVRQA PGKGLEWVSG IYSSGGYTKY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARDL SNGDDVFDIW GQGTMVTVSS 120ASTKGPSVFP LAPSSKS 137 M28-C03 LCQDIQMTQSPS SLSASVGDRV TITCRASQSI NFYLNWYQQK PGKAPKLLIY VASSLESGVP  60SRFSGSASGT EFTLTISSLQ PEDFATYYCL QSYSAPYTFG QGTKVEIT 108 M28-C03 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS YYQMGWVRQA PGKGLEWVSS IYPSGGMTDY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCTRGS PWGDDAFDIW GQGTMVTVSS 120ASTKGPSVFP LAPSSKS 137 M28-D02 LCQDIqMTQSPS SLSASEGDMV TITCRTSRRI GTYLNWYQQK PGKAPKLLIY GASSLQSGVP  60SRFSGSGSGT DFTLTVSSLQ PEDVGTYYCQ QSFSSPWTFG PGTKVEIK 108 M28-D02 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS WYPMQWVRQA PGKGLEWVSY IYPSGGGTDY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TATYYCATSS GWLGDAFDIW GQGTMVTVSS 120ASTKGPSVFP LAPSSKS 137 M28-D12 LCQDIQMTQSPS SLSASVGDRV TITCRASQSI ATYLNWYQQK PGRAPKLLIY AASSLQSGVP  60SRFVGGGSGS GTHFTLTISS LQPEDFATYY CQQSYSTRET FGQGTKVEIK 110 M28-D12 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS WYTMHWVRQA PGKGLEWVSV IYPSGGPTSY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TATYYCARDG SGSYLGFDYW GQGTLVTVSS 120ASTKGPSVFP LAPSSKS 137 M28-E01 LCQDIQMTQSPS SLSASVGDRV TITCRASQSI SSYLNWYQQK PGKAPKLLIY AASSLQSGVP  60SKFSGSGSGT DYTLTISSLQ PEDFATYYCQ QTYTTPWTFG QGTKVEIK 108 M28-E01 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS SYPMGWVRQA PGKGLEWVSR ISSSGGMTIY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARDD WNVGMDVWGQ GTTVTVSSAS 120TKGPSVFPLA PSSKS 135 M28-E11 LCQDIQMTQSPS SVSASVGDRV TINCRASQDI SNWLAWYQQK PGKAPNLLIY AASSLQTGAP  60SRFSGSGSGT DFTLTISSLQ PEDFGTYVCQ QSYSLPWTFG LGTKVEVR 108 M28-E11 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS LYDMTWVRQA PGKGLEWVSG ISSSGGVTIY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARTY YYDSSGYADA FDIWGQGTMV 120TVSSASTKGP SVFPLAPSSK S 141 M28-F01 LCQDIQMTQSPS SLSASVGDRV TITCRASQSI NTYLNWYQQK PGKAPKVLIH AASTLESGVP  60SRFSGSGSGT DFTLTISSLQ PEDFATYYCQ QSYSTPPTFG QGTKVEIK 108 M28-F01 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS VYLMHWVRQA PGKGLEWVSG ISPSGGYTQY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARPG GLDAFDIWGQ GTMVTVSSAS 120TKGPSVFPLA PSSKS 135 M28-F05 LCQDIQMTQSPS SLSASVGDRV TITCRASQSI SSYLNWYQQK PGKAPKLLIY AASSLQSGVP  60SRFSGSGSGT DFTLTISSLQ PEDFATYYCQ QSYSTPLTFG GGTKVEIK 108 M28-F05 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS RYIMWWVRQA PGKGLEWVSG IYSSGGYTQY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAMYYCAREL EGLGGFDYWG QGTLVTVSSA 120STKGPSVFPL APSSKS 136 M28-F07 LCQDIQMTQSPS SVSASVGDRV TITCRASQGI SSWLAWYQQK PGKAPKLLIY ATSGLQSGVP  60SRFSGSGSGT DFTLTISSLQ PEDFATYYCQ QAKSFPLTFG GGTRVEIK 108 M28-F07 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS DYTMYWVRQA PGKGLEWVSS IVPSGGHTLY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAKDH LSSWYGGFFD YWGQGTLVTV 120SSASTKGPSV FPLAPSSKS 139 M29-C07 LCQDIQMTQSPS SLSASVGDRV TITCRASQSI SSYLNWYQQK PGKAPKLLIY AASSLQSGVP  60SRFSGSGSGT DFTLTISSLQ PEDFATYYCQ QSYSTRYTFG QGTKLEIK 108 M29-C07 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS GYDMMWVRQA PGKGLEWVSV ISSSGGNTAY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARES SGLYYFDYWG QGTLVTVSSA 120STKGPSVFPL APSSKS 136 M29-D10 LCQDIQMTQSPS SLSASVGDTV SITCRASQSI TIYLNWYQHK PGKAPNLLIY GASNLHSGVP  60SRFSGSGSGT DFTLTISSLQ PEDFATYYCQ QSYDTPLTFG GGTKVEIK 108 M29-D10 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS WYPMYWVRQA PGKGLEWVSS IGSSGGPTPY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARWA DYGGSLDYWG QGTLVTVSSA 120STKGPSVFPL APSSKS 136 M29-E02 LCQSVLTQPPSV SEAPRQRVTI SCSGSSSNIG NNAVSWYQQL PGKAPKLLIY YDDLLPSGVS  60DRFSGSKSGT SASLAISGLR SEDEADYYCA AWDDSLNGFV FGTGTKVTVL 110 M29-E02 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS RYPMMWVRQA PGKGLEWVSV IYPSGGDTFY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCASGD DYLWEAAVYW GQGTLVTVSS 120ASTKGPSVFP LAPSSKS 137 M29-G08 LCQDIQMTQSPA TLSASPGETV TLSCRASQNI GNDVAWYRQR PGQAPRLLIH HASTRAYGIP  60ARLRGSGSAT EFTLTITSLE PEDFAIYYCQ QFYDWPAHTF ALGTRLEIKR 110 M29-G08 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS YYHMWWVRQA PGKGLEWVSG ISPSGGFTFY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARDY YYDSSGYSPL GYWGQGTLVT 120VSSASTKGPS VFPLAPSSKS 140 M29-G10 LCQDIQMTQSPS SLSSSVGDSA TITCRASQSI SIYLNWYQQK PGKAPKILIY GASQLESGVP  60SRFSGSGSGT DFTLTVSGLQ PEDFATYWCQ QSYNVPYTFG QGTKLEIK 108 M29-G10 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS FYKMIWVRQA PGKGLEWVSS ISSSGGSTQY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARDR VDLGYLDYWG QGTLVTVSSA 120STKGPSVFPL APSSKS 136 M74-A07 LCQDIQMTQSPS SLSASVRDRV TITCRTSQNI NTYLNWYYQA PGRAPKLLIF GVSSLHRGVS  60SRFSGSGDGT EFTLTISSLQ PEDIGTYFCQ QSYSSPWTFG QGTKVEIK 108 M74-A07 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS QYLMMWVRQA PGKGLEWVSS IYPSGGYTSY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARVS TAVADNDYWG QGTLVTVSSA 120STKGPSVFPL APSSKS 136 M76-F02 LCQDIQMTQSPS SLSASVGDRV TITCRASQTI DNYLHWYQQK PGKAPKVLIH DASSLQSGVP  60PRFSGSGSGT DFTLTISSLQ PEDFATYYCQ QSYDTPQYTF GQGTKLEIK 109 M76-F02 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS LYDMNWVRQA PGKGLEWVSG ISPSGGQTMY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARQP MISAFDIWGQ GTMVTVSSAS 120TKGPSVFPLA PSSKS 135 M76-G02 LCQDIQMTQSPS SLSASVGDRV TITCRASQSI SSYLNWYQQK PGKAPKLLIY AASSLQSGVP  60SRFSGSGSGT DFTLTISSLQ PEDFATYYCQ QSYSTPPWTF GQGTKVEIK 109 M76-G02 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS LYAMWWVRQA PGKGLEWVSY ISSSGGFTSY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARYR VGVAATDYWG QGTLVTVSSA 120STKGPSVFPL APSSKS 136 M76-G06 LCQDIQMTQSPS SLSASVRDRV TITCRASQSI STYLNWYQQK PGEAPKLLVF AASSLQSGVP  60SRFSGSGSGT DFTLSISSLQ PEDFATYYCQ QSYSTPHTFG QGAKVEIK 108 M76-G06 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS GYIMHWVRQA PGKGLEWVSW IYPSGGWTEY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARDA PGVGAIDYWG QGTLVTVSSA 120STKGPSVFPL APSSKS 136 M76-H02 LCQDIQMTQSPS SLSASEGDRV TITCRASQDI SVYLNWYQMK SGKAPKLLIY GGASLQSGVP  60ARFSGSGYGT DFTLTITDLR PEDFATYYCQ QSYSLPFTFG GGTKVEIK 108 M76-H02 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS MYWMQWVRQA PGKGLEWVSY IYPSGGPTKY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARPS GSYGDAFDIW GQGTMVTVSS 120ASTKGPSVFP LAPSSKS 137 M77-C07 LCQDIQMTQSPS TLSASVGDRV TITCRASQNI SSYLNWYQQK PGKAPKLLIY AASSLQSGVP  60SRFSGSGSGT DFTLTISSLQ PEDFATYSCQ QSYSTPRTFG QGTKVEIK 108 M77-C07 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS LYIMGWVRQA PGKGLEWVSG IYPSGGFTMY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARES SGVAAPDYWG QGTLVTVSSA 120STKGPSVFPL APSSKS 136 M77-H04 LCQDIQMTQSPL SLPVTPGEPA SISCRSSQSL LHSRGYNYLD WYLQKPGQSP QLLIYLGSNR  60ASGVPDRFSG SGSGTDFTLK ISRVEAEDVG VYYCMQALQR RTFGQGTKLE IK 112 M77-H04 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS YYTMIWVRQA PGKGLEWVSG IRSSGGGTRY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAKDG SRYSYGSIYY YYGMDAWGQG 120TTVTVSSAST KGPSVFPLAP SSKS 144

Example 2: Lead Antibody Inhibitors

Antibodies were selected as lead plasma kallikrein inhibitors on thebasis of apparent inhibition constant (K_(i,app)), specificity withrespect to lack of inhibition of other serine proteases, inhibition ofbradykinin generation, and lack of binding to plasma prekallikrein(Table 3). Plasma kallikrein circulates in the plasma as an inactivezymogen (prekallikrein) at a concentration of approximately 500 nM.Antibodies that bound prekallikrein may be rendered inaccessible towardsactive plasma kallikrein inhibition and could substantially increase thein vivo dose required for efficacy. Therefore, a surface plasmonresonance (SPR) assay was used to identify antibodies that do not bindprekallikrein (data not shown). Specifically, human IgGs (X81-B01,M162-A04 (R84-H05); M160-G12 (R84-D02); and M142-H08) were captured on aCM5 chip using an anti-human Fc surface and 100 nM of plasma kallikreinor 100 nM or 500 nM prekallikrein. The prekallikrein was treated withaprotinin-sepharose to remove active plasma kallikrein. Theprekallikrein used for X81-B01 was buffer exchanged into the exactpreparation of SPR running buffer (HEPES buffered saline) to avoid therefractive index shift that was observed with three other antibodiesthat were tested: M162-A04 (R84-H05); M160-G12 (R84-D02); and M142-H08.

Of the antibodies listed in Table 3, only M142-H08 inhibits human plasmakallikrein with a subnanomolar K_(i,app). However, when M142-H08 wasproduced as an IgG it was found to be cleaved in the CDR3 of the heavychain. Consequently, we decided to undertake two approaches to improvethe affinity: 1) affinity maturation of M162-A04 and M160-G12 using anovel form of light chain shuffling called ROLIC (Rapid Optimization ofLight Chains) (see, e.g., WO 2009/102927 and U.S. 2009-0215119); and 2)sequence optimization of M142-H08 in order to prevent the cleavage ofthe IgG that occurs while retaining the binding and inhibitor propertiesof M142-H08.

TABLE 3 Top Ranking Antibody Inhibitors of PKal Before AffinityMaturation or Sequence Optimization Criteria M162-A04 M160-G12M142-H08^(a) K_(i,app) human pKal 2 nM (as an 5.6 nM (as an 0.6 nM (as aIgG) IgG) Fab) K_(i,app) rodent pKal 2 nM (mouse <1 nM ~1 nM (mouse andrat) (mouse) and rat) Binds prekallikrein? No No No Specific inhibitorwith Yes Yes Yes respect to fXIa, plasmin, and trypsin Inhibitsbradykinin Yes Yes Yes generation ^(a)When M142-H08 was produced as anIgG it was determined to be cleaved in the CDR3 of its heavy chain(GGLLLWFR-ELKSNYFDY (SEQ ID NO: 894)).

Example 3: Sequence Optimization of M142-H08

Of the antibodies listed in Table 3, only M142-H08 inhibits human pKalwith a subnanomolar K_(i,app). However, when M142-H08 was produced as anIgG it was found to be cleaved in the CDR3 of the heavy chain. M142-H08was found by mass spectrometry to be cleaved after the arginine in the“WFR” sequence of the HC-CDR3 sequence (GGLLLWFRELKSNYFDY (SEQ IDNO:894)). This cleavage suggests that a protease from the cells used toexpress the antibody (both CHO and 293T human kidney cells) isenzymatically cleaving the antibody at a single specific site. Wemutated the HC-CDR3 sequence of M142-H08 in order to identify amino acidsubstitutions that prevent the cleavage of the IgG that occurs whileretaining the binding and inhibitor properties of M142-H08. Previousexperience with similarly “clipped” antibodies suggested that focusingsimply on the putative P1 position (protease subsite 1, see Table 4) maynot be sufficient to identify antibodies that retain potent inhibitionof the target enzyme while not being clipped by a host cell protease.Therefore, we created a small library of single point mutations in theregion around the cleavage site in order to identify variants ofM142-H08 that are not clipped but are still potent pKal inhibitors. Werefer to this library as the “CDR3 by Design” library. The small librarywas constructed using a PCR primer that contains the randomized codonNNK at either the P3, the P2, the P1, or the P1′ site. This results in asmall library where each of the 4 positions may contain any of the 20amino acids (20+20+20+20=80 members). Using PCR, this library was clonedinto the M142-H08 Fab sequence in the pMid21 vector, which is a standardphagemid vector.

TABLE 4 Primer sequences Primer Name Sequence N (SEQ ID              P3 P2 P1 P1′ P2′ NO: 895) G  G  L  L  L  W  F  R  E  L  KS N Y 559A.P1.top GGC GGT CTA TTA CTA TGG TTC NNK  20 (SEQ IDGAG CTG AAG TCT AAC TAC NO: 896) 559A.P2.topGGC GGT CTA TTA CTA TGG NNK AGG  20 (SEQ ID GAG CTG AAG TCT AAC TACNO: 897) 559A.P3.top GGC GGT CTA TTA CTA NNK TTC AGG  20 (SEQ IDGAG CTG AAG TCT AAC TAC NO: 898) 559A.P1p.topGGC GGT CTA TTA CTA TGG TTC AGG  20 (SEQ ID NNK CTG AAG TCT AAC TACNO: 899)

By DNA sequencing, we recovered 61 of the possible 80 antibodies (Table5). These antibodies were produced as Fab fragments in small scale (˜20μg) and tested for inhibition against human pKal in an in vitro proteasecleavage assay using Pro-Phe-Arg-aminomethylcoumarin as the syntheticpeptide substrate. The Fabs that were found to be inhibitors of humanpKal were subcloned into our pBRH1f vector (a vector for transientexpression of IgGs in 293T cells) for conversion to full length humanIgG1 antibodies. Five antibodies were then expressed in 293T cells andpurified by protein A sepharose chromatography. The antibodies wereanalyzed by SDS-PAGE to determine which of the inhibitory mutants arenot cleaved by the host cell protease(s) (data not shown). The cleavedantibodies (559A-X67-G05, 559A-X67-H01, 559A-X67-G09) had an extra bandthat migrated between the 38 and the 49 kDa molecular weight marker.This band is absent in the 559A-X67-H04 and 559A-X67-D03 antibodies,which indicates that these antibodies are intact.

K_(i,app) values were determined by steady state enzyme kinetics forthose that were shown by SDS-PAGE to be not cleaved (Table 5).Interestingly, the P2 position was the only position where amino acidsubstitutions yielded intact antibody inhibitors of pKal. Of the 14different mutations that were recovered at the P3 position (Table 5),only one mutant (W to L) was found to be a pKal inhibitor as a Fab butit was subsequently shown to be clipped as an IgG. None of the 16different mutations at the P1 position (Table 5) were found to be pKalinhibitors. Eight of the 15 different mutations at the P1′ position werefound to be inhibitors of pKal as a Fab but all were clipped as an IgG.Consequently, only mutations at the P2 position led to antibodyinhibitors that were not clipped during expression. Of the 16 differentmutations that were recovered at the P2 position (Table 5), eightmutants were found to be a pKal inhibitor as a Fab but it wassubsequently shown to be clipped as an IgG. Four mutants at the P2position were found to have subnanomolar K_(i,app) values: X67-G04 (F toA), X67-C03 (F to M), X67-F01 (F to Q) and X67-D03 (F to N). Theantibody with the highest potency is X67-D03 (K_(i,app)=0.1 nM). The twoantibodies shown in Table 6 were not cleaved when expressed as IgGs andwere found to inhibit pKal with a subnanomolar K_(i,app).

DNA and amino acid sequence alignments of the light chains ofnongermlined (X63-G06) and germlined, codon optimized (X81-B01) versionsof the same antibody discovered using ROLIC affinity maturation areshown in FIGS. 4 and 5, respectively. DNA and amino acid sequencealignments of the heavy chains of nongermlined (X63-G06) and germlined,codon optimized (X81-B01) versions of the same antibody discovered usingROLIC affinity maturation are shown in FIGS. 6 and 7, respectively.

TABLE 5 HV-CDR3 Sequences Obtained from “CDR3 by   Design” Library* Ki,Inhi- In- app Muta- Anti- HV-CDR3 bit tact as an tion body (SEQ ID NOs:as a as an IgG Site I.D. 900-961) Fab? IgG? (nM) Parental X69-C09GGLLLWFRELKSNYFDY Yes No 0.2 P3 X68-E07 GGLLLAFRELKSNYFDY No n/a n/a P3X68-E12 GGLLLCFRELKSNYFDY No n/a n/a P3 X68-A03 GGLLLDFRELKSNYFDY No n/an/a P3 X68-E03 GGLLLEFRELKSNYFDY No n/a n/a P3 X68-A12 GGLLLGFRELKSNYFDYNo n/a n/a P3 X68-D11 GGLLLKFRELKSNYFDY No n/a n/a P3 X68-E01GGLLLLFRELKSNYFDY Yes No n/a P3 X68-F05 GGLLLMFRELKSNYFDY No n/a n/a P3X68-D10 GGLLLPFRELKSNYFDY No n/a n/a P3 X68-F10 GGLLLQFRELKSNYFDY No n/an/a P3 X68-G01 GGLLLRFRELKSNYFDY No n/a n/a P3 X68-G05 GGLLLSFRELKSNYFDYNo n/a n/a P3 X68-F12 GGLLLTFRELKSNYFDY No n/a n/a P3 X68-H04GGLLLVFRELKSNYFDY No n/a n/a P2 X67-G04 GGLLLWARELKSNYFDY Yes Yes 0.35P2 X67-G01 GGLLLWCRELKSNYFDY No n/a n/a P2 X67-E04 GGLLLWDRELKSNYFDY YesYes 1.3 P2 X67-H04 GGLLLWERELKSNYFDY Yes Yes 3.6 P2 X67-C09GGLLLWGRELKSNYFDY Yes Yes 8.6 P2 X67-B04 GGLLLWKRELKSNYFDY Yes No n/a P2X67-G09 GGLLLWLRELKSNYFDY Yes No n/a P2 X67-C03 GGLLLWMRELKSNYFDY YesYes 0.7 P2 X67-D03 GGLLLWNRELKSNYFDY Yes Yes 0.1 P2 X67-B05GGLLLWPRELKSNYFDY No n/a n/a P2 X67-F01 GGLLLWQRELKSNYFDY Yes Yes 0.9 P2X67-G05 GGLLLWRRELKSNYFDY Yes No n/a P2 X67-B03 GGLLLWSRELKSNYFDY YesYes 2.1 P2 X67-F10 GGLLLWTRELKSNYFDY Yes Yes 1.3 P2 X67-H01GGLLLWWRELKSNYFDY Yes No n/a P2 X67-F08 GGLLLWYRELKSNYFDY Yes No n/a P1X66-E09 GGLLLWFAELKSNYFDY No n/a n/a P1 X66-B05 GGLLLWFCELKSNYFDY No n/an/a P1 X66-D03 GGLLLWFEELKSNYFDY No n/a n/a P1 X66-H04 GGLLLWFFELKSNYFDYNo n/a n/a P1 X66-H02 GGLLLWFGELKSNYFDY No n/a n/a P1 X66-C11GGLLLWFHELKSNYFDY No n/a n/a P1 X66-A07 GGLLLWFKELKSNYFDY No n/a n/a P1X66-C03 GGLLLWFLELKSNYFDY No n/a n/a P1 X66-G05 GGLLLWFMELKSNYFDY No n/an/a P1 X66-F10 GGLLLWFPELKSNYFDY No n/a n/a P1 X66-E04 GGLLLWFQELKSNYFDYNo n/a n/a P1 X66-F01 GGLLLWFSELKSNYFDY No n/a n/a P1 X66-H11GGLLLWFTELKSNYFDY No n/a n/a P1 X66-C02 GGLLLWFVELKSNYFDY No n/a n/a P1X66-F09 GGLLLWFWELKSNYFDY No n/a n/a P1 X66-G08 GGLLLWFYELKSNYFDY No n/an/a P1′ X69-D08 GGLLLWFRALKSNYFDY No n/a n/a P1′ X69-B02GGLLLWFRCLKSNYFDY No n/a n/a P1′ X69-D09 GGLLLWFRGLKSNYFDY Yes No n/aP1′ X69-D02 GGLLLWFRHLKSNYFDY No n/a n/a P1′ X69-A12 GGLLLWFRKLKSNYFDYNo n/a n/a P1′ X69-F05 GGLLLWFRLLKSNYFDY Yes No n/a P1′ X69-B08GGLLLWFRNLKSNYFDY Yes No n/a P1′ X69-A10 GGLLLWFRPLKSNYFDY No n/a n/aP1′ X69-A09 GGLLLWFRQLKSNYFDY Yes No n/a P1′ X69-E05 GGLLLWFRRLKSNYFDYNo n/a n/a P1′ X69-F09 GGLLLWFRSLKSNYFDY Yes No n/a P1′ X69-F01GGLLLWFRTLKSNYFDY Yes No n/a P1′ X69-C12 GGLLLWFRVLKSNYFDY Yes No n/aP1′ X69-E01 GGLLLWFRWLKSNYFDY Yes No n/a P1′ X69-H10 GGLLLWFRYLKSNYFDYNo n/a n/a *All of these antibodies are single point mutations of theM142-H08 sequence.

Amino acid sequences of light chain (LC) and heavy chain (HC) variabledomain of pKal antibodies with designed HC CDR3s are shown below. (SEQID NOs: 962-1085)

X68-E07 LCQDIQMTQSPS SLSAFVGDRV TITCRASQPI DNYLNWYHQK PGKAPKLLIY AASRLQSGVP  60SRLSGSGFGT DFTLTISSLQ PEDFGNYYCQ QSYTVPYTFG GGTKVEIR 108 X68-E07 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS AYSMIWVRQA PGKGLEWVSY IRPSGGRTTY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARGG LLLAFRELKS NYFDYWGQGT 120LVTVSSASTK GPSVFPLAPS SKS 143 X68-E12 LCQDIQMTQSPS SLSAFVGDRV TITCRASQPI DNYLNWYHQK PGKAPKLLIY AASRLQSGVP  60SRLSGSGFGT DFTLTISSLQ PEDFGNYYCQ QSYTVPYTFG GGTKVEIR 108 X68-E12 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS AYSMIWVRQA PGKGLEWVSY IRPSGGRTTY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARGG LLLCFRELKS NYFDYWGQGT 120LVTVSSASTK GPSVFPLAPS SKS 143 X68-A03 LCQDIQMTQSPS SLSAFVGDRV TITCRASQPI DNYLNWYHQK PGKAPKLLIY AASRLQSGVP  60SRLSGSGFGT DFTLTISSLQ PEDFGNYYCQ QSYTVPYTFG GGTKVEIR 108 X68-A03 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS AYSMIWVRQA PGKGLEWVSY IRPSGGRTTY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARGG LLLDFRELKS NYFDYWGQGT 120LVTVSSASTK GPSVFPLAPS SKS 143 X68-E03 LCQDIQMTQSPS SLSAFVGDRV TITCRASQPI DNYLNWYHQK PGKAPKLLIY AASRLQSGVP  60SRLSGSGFGT DFTLTISSLQ PEDFGNYYCQ QSYTVPYTFG GGTKVEIR 108 X68-E03 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS AYSMIWVRQA PGKGLEWVSY IRPSGGRTTY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARGG LLLEFRELKS NYFDYWGQGT 120LVTVSSASTK GPSVFPLAPS SKS 143 X68-A12 LCQDIQMTQSPS SLSAFVGDRV TITCRASQPI DNYLNWYHQK PGKAPKLLIY AASRLQSGVP  60SRLSGSGFGT DFTLTISSLQ PEDFGNYYCQ QSYTVPYTFG GGTKVEIR 108 X68-A12 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS AYSMIWVRQA PGKGLEWVSY IRPSGGRTTY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARGG LLLGFRELKS NYFDYWGQGT 120LVTVSSASTK GPSVFPLAPS SKS 143 X68-D11 LCQDIQMTQSPS SLSAFVGDRV TITCRASQPI DNYLNWYHQK PGKAPKLLIY AASRLQSGVP  60SRLSGSGFGT DFTLTISSLQ PEDFGNYYCQ QSYTVPYTFG GGTKVEIR 108 X68-D11 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS AYSMIWVRQA PGKGLEWVSY IRPSGGRTTY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARGG LLLKFRELKS NYFDYWGQGT 120LVTVSSASTK GPSVFPLAPS SKS 143 X68-E01 LCQDIQMTQSPS SLSAFVGDRV TITCRASQPI DNYLNWYHQK PGKAPKLLIY AASRLQSGVP  60SRLSGSGFGT DFTLTISSLQ PEDFGNYYCQ QSYTVPYTFG GGTKVEIR 108 X68-E01 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS AYSMIWVRQA PGKGLEWVSY IRPSGGRTTY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARGG LLLLFRELKS NYFDYWGQGT 120LVTVSSASTK GPSVFPLAPS SKS 143 X68-F05 LCQDIQMTQSPS SLSAFVGDRV TITCRASQPI DNYLNWYHQK PGKAPKLLIY AASRLQSGVP  60SRLSGSGFGT DFTLTISSLQ PEDFGNYYCQ QSYTVPYTFG GGTKVEIR 108 X68-F05 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS AYSMIWVRQA PGKGLEWVSY IRPSGGRTTY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARGG LLLMFRELKS NYFDYWGQGT 120LVTVSSASTK GPSVFPLAPS SKS 143 X68-D10 LCQDIQMTQSPS SLSAFVGDRV TITCRASQPI DNYLNWYHQK PGKAPKLLIY AASRLQSGVP  60SRLSGSGFGT DFTLTISSLQ PEDFGNYYCQ QSYTVPYTFG GGTKVEIR 108 X68-D10 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS AYSMIWVRQA PGKGLEWVSY IRPSGGRTTY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARGG LLLPFRELKS NYFDYWGQGT 120LVTVSSASTK GPSVFPLAPS SKS 143 X68-F10 LCQDIQMTQSPS SLSAFVGDRV TITCRASQPI DNYLNWYHQK PGKAPKLLIY AASRLQSGVP  60SRLSGSGFGT DFTLTISSLQ PEDFGNYYCQ QSYTVPYTFG GGTKVEIR 108 X68-F10 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS AYSMIWVRQA PGKGLEWVSY IRPSGGRTTY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARGG LLLQFRELKS NYFDYWGQGT 120LVTVSSASTK GPSVFPLAPS SKS 143 X68-G01 LCQDIQMTQSPS SLSAFVGDRV TITCRASQPI DNYLNWYHQK PGKAPKLLIY AASRLQSGVP  60SRLSGSGFGT DFTLTISSLQ PEDFGNYYCQ QSYTVPYTFG GGTKVEIR 108 X68-G01 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS AYSMIWVRQA PGKGLEWVSY IRPSGGRTTY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARGG LLLRFRELKS NYFDYWGQGT 120LVTVSSASTK GPSVFPLAPS SKS 143 X68-G05 LCQDIQMTQSPS SLSAFVGDRV TITCRASQPI DNYLNWYHQK PGKAPKLLIY AASRLQSGVP  60SRLSGSGFGT DFTLTISSLQ PEDFGNYYCQ QSYTVPYTFG GGTKVEIR 108 X68-G05 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS AYSMIWVRQA PGKGLEWVSY IRPSGGRTTY  60ADSVKGRFTI SRDNSKNILY LQMNSLRAED TAVYYCARGG LLLSFRELKS NYFDYWGQGT 120LVTVSSASTK GPSVFPLAPS SKS 143 X68-F12 LCQDIQMTQSPS SLSAFVGDRV TITCRASQPI DNYLNWYHQK PGKAPKLLIY AASRLQSGVP  60SRLSGSGFGT DFTLTISSLQ PEDFGNYYCQ QSYTVPYTFG GGTKVEIR 108 X68-F12 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS AYSMIWVRQA PGKGLEWVSY IRPSGGRTTY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARGG LLLTFRELKS NYFDYWGQGT 120LVTVSSASTK GPSVFPLAPS SKS 143 X68-H04 LCQDIQMTQSPS SLSAFVGDRV TITCRASQPI DNYLNWYHQK PGKAPKLLIY AASRLQSGVP  60SRLSGSGFGT DFTLTISSLQ PEDFGNYYCQ QSYTVPYTFG GGTKVEIR 108 X68-H04 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS AYSMIWVRQA PGKGLEWVSY IRPSGGRTTY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARGG LLLVFRELKS NYFDYWGQGT 120LVTVSSASTK GPSVFPLAPS SKS 143 X67-G04 LCQDIQMTQSPS SLSAFVGDRV TITCRASQPI DNYLNWYHQK PGKAPKLLIY AASRLQSGVP  60SRLSGSGFGT DFTLTISSLQ PEDFGNYYCQ QSYTVPYTFG GGTKVEIR 108 X67-G04 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS AYSMIWVRQA PGKGLEWVSY IRPSGGRTTY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARGG LLLWARELKS NYFDYWGQGT 120LVTVSSASTK GPSVFPLAPS SKS 143 X67-G01 LCQDIQMTQSPS SLSAFVGDRV TITCRASQPI DNYLNWYHQK PGKAPKLLIY AASRLQSGVP  60SRLSGSGFGT DFTLTISSLQ PEDFGNYYCQ QSYTVPYTFG GGTKVEIR 108 X67-G01 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS AYSMIWVRQA PGKGLEWVSY IRPSGGRTTY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARGG LLLWCRELKS NYFDYWGQGT 120LVTVSSASTK GPSVFPLAPS SKS 143 X67-E04 LCQDIQMTQSPS SLSAFVGDRV TITCRASQPI DNYLNWYHQK PGKAPKLLIY AASRLQSGVP  60SRLSGSGFGT DFTLTISSLQ PEDFGNYYCQ QSYTVPYTFG GGTKVEIR 108 X67-E04 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS AYSMIWVRQA PGKGLEWVSY IRPSGGRTTY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARGG LLLWDRELKS NYFDYWGQGT 120LVTVSSASTK GPSVFPLAPS SKS 143 X67-H04 LCQDIQMTQSPS SLSAFVGDRV TITCRASQPI DNYLNWYHQK PGKAPKLLIY AASRLQSGVP  60SRLSGSGFGT DFTLTISSLQ PEDFGNYYCQ QSYTVPYTFG GGTKVEIR 108 X67-H04 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS AYSMIWVRQA PGKGLEWVSY IRPSGGRTTY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARGG LLLWERELKS NYFDYWGQGT 120LVTVSSASTK GPSVFPLAPS SKS 143 X66-E09 LCQDIQMTQSPS SLSAFVGDRV TITCRASQPI DNYLNWYHQK PGKAPKLLIY AASRLQSGVP  60SRLSGSGFGT DFTLTISSLQ PEDFGNYYCQ QSYTVPYTFG GGTKVEIR 108 X66-E09 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS AYSMIWVRQA PGKGLEWVSY IRPSGGRTTY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARGG LLLWFAELKS NYFDYWGQGT 120LVTVSSASTK GPSVFPLAPS SKS 143 X66-B05 LCQDIQMTQSPS SLSAFVGDRV TITCRASQPI DNYLNWYHQK PGKAPKLLIY AASRLQSGVP  60SRLSGSGFGT DFTLTISSLQ PEDFGNYYCQ QSYTVPYTFG GGTKVEIR 108 X66-B05 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS AYSMIWVRQA PGKGLEWVSY IRPSGGRTTY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARGG LLLWFCELKS NYFDYWGQGT 120LVTVSSASTK GPSVFPLAPS SKS 143 X66-D03 LCQDIQMTQSPS SLSAFVGDRV TITCRASQPI DNYLNWYHQK PGKAPKLLIY AASRLQSGVP  60SRLSGSGFGT DFTLTISSLQ PEDFGNYYCQ QSYTVPYTFG GGTKVEIR 108 X66-D03 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS AYSMIWVRQA PGKGLEWVSY IRPSGGRTTY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARGG LLLWFEELKS NYFDYWGQGT 120LVTVSSASTK GPSVFPLAPS SKS 143 X66-H04 LCQDIQMTQSPS SLSAFVGDRV TITCRASQPI DNYLNWYHQK PGKAPKLLIY AASRLQSGVP  60SRLSGSGFGT DFTLTISSLQ PEDFGNYYCQ QSYTVPYTFG GGTKVEIR 108 X66-H04 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS AYSMIWVRQA PGKGLEWVSY IRPSGGRTTY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARGG LLLWFFELKS NYFDYWGQGT 120LVTVSSASTK GPSVFPLAPS SKS 143 X66-H02 LCQDIQMTQSPS SLSAFVGDRV TITCRASQPI DNYLNWYHQK PGKAPKLLIY AASRLQSGVP  60SRLSGSGFGT DFTLTISSLQ PEDFGNYYCQ QSYTVPYTFG GGTKVEIR 108 X66-H02 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS AYSMIWVRQA PGKGLEWVSY IRPSGGRTTY  60ADSVKGRFTI SRDNSKNTLY LQMHSLRAED TAVYYCARGG LLLWFGELKS NYFDYWGQGT 120LVTVSSASTK GPSVFPLAPS SKS 143 X66-C11 LCQDIQMTQSPS SLSAFVGDRV TITCRASQPI DNYLNWYHQK PGKAPKLLIY AASRLQSGVP  60SRLSGSGFGT DFTLTISSLQ PEDFGNYYCQ QSYTVPYTFG GGTKVEIR 108 X66-C11 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS AYSMIWVRQA PGKGLEWVSY IRPSGGRTTY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARGG LLLWFHELKS NYFDYWGQGT 120LVTVSSASTK GPSVFPLAPS SKS 143 X66-A07 LCQDIQMTQSPS SLSAFVGDRV TITCRASQPI DNYLNWYHQK PGKAPKLLIY AASRLQSGVP  60SRLSGSGFGT DFTLTISSLQ PEDFGNYYCQ QSYTVPYTFG GGTKVEIR 108 X66-A07 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS AYSMIWVRQA PGKGLEWVSY IRPSGGRTTY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARGG LLLWFKELKS NYFDYWGQGT 120LVTVSSASTK GPSVFPLAPS SKS 143 X66-C03 LCQDIQMTQSPS SLSAFVGDRV TITCRASQPI DNYLNWYHQK PGKAPKLLIY AASRLQSGVP  60SRLSGSGFGT DFTLTISSLQ PEDFGNYYCQ QSYTVPYTFG GGTKVEIR 108 X66-C03 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS AYSMIWVRQA PGKGLEWVSY IRPSGGRTTY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARGG LLLWFLELKS NYFDYWGQGT 120LVTVSSASTK GPSVFPLAPS SKS 143 X66-G05 LCQDIQMTQSPS SLSAFVGDRV TITCRASQPI DNYLNWYHQK PGKAPKLLIY AASRLQSGVP  60SRLSGSGFGT DFTLTISSLQ PEDFGNYYCQ QSYTVPYTFG GGTKVEIR 108 X66-G05 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS AYSMIWVRQA PGKGLEWVSY IRPSGGRTTY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARGG LLLWFMELKS NYFDYWGQGT 120LVTVSSASTK GPSVFPLAPS SKS 143 X66-F10 LCQDIQMTQSPS SLSAFVGDRV TITCRASQPI DNYLNWYHQK PGKAPKLLIY AASRLQSGVP  60SRLSGSGFGT DFTLTISSLQ PEDFGNYYCQ QSYTVPYTFG GGTKVEIR 108 X66-F10 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS AYSMIWVRQA PGKGLEWVSY IRPSGGRTTY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARGG LLLWFPELKS NYFDYWGQGT 120LVTVSSASTK GPSVFPLAPS SKS 143 X66-E04 LCQDIQMTQSPS SLSAFVGDRV TITCRASQPI DNYLNWYHQK PGKAPKLLIY AASRLQSGVP  60SRLSGSGFGT DFTLTISSLQ PEDFGNYYCQ QSYTVPYTFG GGTKVEIR 108 X66-E04 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS AYSMIWVRQA PGKGLEWVSY IRPSGGRTTY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARGG LLLWFQELKS NYFDYWGQGT 120LVTVSSASTK GPSVFPLAPS SKS 143 X69-D08 LCQDIQMTQSPS SLSAFVGDRV TITCRASQPI DNYLNWYHQK PGKAPKLLIY AASRLQSGVP  60SRLSGSGFGT DFTLTISSLQ PEDFGNYYCQ QSYTVPYTFG GGTKVEIR 108 X69-D08 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS AYSMIWVRQA PGKGLEWVSY IRPSGGRTTY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARGG LLLWFRALKS NYFDYWGQGT 120LVTVSSASTK GPSVFPLAPS SKS 143 X69-B02 LCQDIQMTQSPS SLSAFVGDRV TITCRASQPI DNYLNWYHQK PGKAPKLLIY AASRLQSGVP  60SRLSGSGFGT DFTLTISSLQ PEDFGNYYCQ QSYTVPYTFG GGTKVEIR 108 X69-B02 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS AYSMIWVRQA PGKGLEWVSY IRPSGGRTTY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARGG LLLWFRCLKS NYFDYWGQGT 120LVTVSSASTK GPSVFPLAPS SKS 143 X69-C09 LCQDIQMTQSPS SLSAFVGDRV TITCRASQPI DNYLNWYHQK PGKAPKLLIY AASRLQSGVP  60SRLSGSGFGT DFTLTISSLQ PEDFGNYYCQ QSYTVPYTFG GGTKVEIR 108 X69-C09 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS AYSMIWVRQA PGKGLEWVSY IRPSGGRTTY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARGG LLLWFRELKS NYFDYWGQGT 120LVTVSSASTK GPSVFPLAPS SKS 143 X69-D09 LCQDIQMTQSPS SLSAFVGDRV TITCRASQPI DNYLNWYHQK PGKAPKLLIY AASRLQSGVP  60SRLSGSGFGT DFTLTISSLQ PEDFGNYYCQ QSYTVPYTFG GGTKVEIR 108 X69-D09 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS AYSMIWVRQA PGKGLEWVSY IRPSGGRTTY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARGG LLLWFRGLKS NYFDYWGQGT 120LVTVSSASTK GPSVFPLAPS SKS 143 X69-D02 LCQDIQMTQSPS SLSAFVGDRV TITCRASQPI DNYLNWYHQK PGKAPKLLIY AASRLQSGVP  60SRLSGSGFGT DFTLTISSLQ PEDFGNYYCQ QSYTVPYTFG GGTKVEIR 108 X69-D02 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS AYSMIWVRQA PGKGLEWVSY IRPSGGRTTY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARGG LLLWFRHLKS NYFDYWGQGT 120LVTVSSASTK GPSVFPLAPS SKS 143 X69-A12 LCQDIQMTQSPS SLSAFVGDRV TITCRASQPI DNYLNWYHQK PGKAPKLLIY AASRLQSGVP  60SRLSGSGFGT DFTLTISSLQ PEDFGNYYCQ QSYTVPYTFG GGTKVEIR 108 X69-A12 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS AYSMIWVRQA PGKGLEWVSY IRPSGGRTTY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARGG LLLWFRKLKS NYFDYWGQGT 120LVTVSSASTK GPSVFPLAPS SKS 143 X69-F05 LCQDIQMTQSPS SLSAFVGDRV TITCRASQPI DNYLNWYHQK PGKAPKLLIY AASRLQSGVP  60SRLSGSGFGT DFTLTISSLQ PEDFGNYYCQ QSYTVPYTFG GGTKVEIR 108 X69-F05 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS AYSMIWVRQA PGKGLEWVSY IRPSGGRTTY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARGG LLLWFRLLKS NYFDYWGQGT 120LVTVSSASTK GPSVFPLAPS SKS 143 X69-B08 LCQDIQMTQSPS SLSAFVGDRV TITCRASQPI DNYLNWYHQK PGKAPKLLIY AASRLQSGVP  60SRLSGSGFGT DFTLTISSLQ PEDFGNYYCQ QSYTVPYTFG GGTKVEIR 108 X69-B08 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS AYSMIWVRQA PGKGLEWVSY IRPSGGRTTY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARGG LLLWFRNLKS NYFDYWGQGT 120LVTVSSASTK GPSVFPLAPS SKS 143 X69-A10 LCQDIQMTQSPS SLSAFVGDRV TITCRASQPI DNYLNWYHQK PGKAPKLLIY AASRLQSGVP  60SRLSGSGFGT DFTLTISSLQ PEDFGNYYCQ QSYTVPYTFG GGTKVEIR 108 X69-A10 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS AYSMIWVRQA PGKGLEWVSY IRPSGGRTTY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARGG LLLWFRPLKS NYFDYWGQGT 120LVTVSSASTK GPSVFPLAPS SKS 143 X69-A09 LCQDIQMTQSPS SLSAFVGDRV TITCRASQPI DNYLNWYHQK PGKAPKLLIY AASRLQSGVP  60SRLSGSGFGT DFTLTISSLQ PEDFGNYYCQ QSYTVPYTFG GGTKVEIR 108 X69-A09 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS AYSMIWVRQA PGKGLEWVSY IRPSGGRTTY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARGG LLLWFRQLKS NYFDYWGQGT 120LVTVSSASTK GPSVFPLAPS SKS 143 X69-E05 LCQDIQMTQSPS SLSAFVGDRV TITCRASQPI DNYLNWYHQK PGKAPKLLIY AASRLQSGVP  60SRLSGSGFGT DFTLTISSLQ PEDFGNYYCQ QSYTVPYTFG GGTKVEIR 108 X69-E05 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS AYSMIWVRQA PGKGLEWVSY IRPSGGRTTY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARGG LLLWFRRLKS NYFDYWGQGT 120LVTVSSASTK GPSVFPLAPS SKS 143 X69-F09 LCQDIQMTQSPS SLSAFVGDRV TITCRASQPI DNYLNWYHQK PGKAPKLLIY AASRLQSGVP  60SRLSGSGFGT DFTLTISSLQ PEDFGNYYCQ QSYTVPYTFG GGTKVEIR 108 X69-F09 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS AYSMIWVRQA PGKGLEWVSY IRPSGGRTTY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARGG LLLWFRSLKS NYFDYWGQGT 120LVTVSSASTK GPSVFPLAPS SKS 143 X69-F01 LCQDIQMTQSPS SLSAFVGDRV TITCRASQPI DNYLNWYHQK PGKAPKLLIY AASRLQSGVP  60SRLSGSGFGT DFTLTISSLQ PEDFGNYYCQ QSYTVPYTFG GGTKVEIR 108 X69-F01 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS AYSMIWVRQA PGKGLEWVSY IRPSGGRTTY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARGG LLLWFRTLKS NYFDYWGQGT 120LVTVSSASTK GPSVFPLAPS SKS 143 X69-C12 LCQDIQMTQSPS SLSAFVGDRV TITCRASQPI DNYLNWYHQK PGKAPKLLIY AASRLQSGVP  60SRLSGSGFGT DFTLTISSLQ PEDFGNYYCQ QSYTVPYTFG GGTKVEIR 108 X69-C12 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS AYSMIWVRQA PGKGLEWVSY IRPSGGRTTY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARGG LLLWFRVLKS NYFDYWGQGT 120LVTVSSASTK GPSVFPLAPS SKS 143 X69-E01 LCQDIQMTQSPS SLSAFVGDRV TITCRASQPI DNYLNWYHQK PGKAPKLLIY AASRLQSGVP  60SRLSGSGFGT DFTLTISSLQ PEDFGNYYCQ QSYTVPYTFG GGTKVEIR 108 X69-E01 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS AYSMIWVRQA PGKGLEWVSY IRPSGGRTTY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARGG LLLWFRWLKS NYFDYWGQGT 120LVTVSSASTK GPSVFPLAPS SKS 143 X69-H10 LCQDIQMTQSPS SLSAFVGDRV TITCRASQPI DNYLNWYHQK PGKAPKLLIY AASRLQSGVP  60SRLSGSGFGT DFTLTISSLQ PEDFGNYYCQ QSYTVPYTFG GGTKVEIR 108 X69-H10 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS AYSMIWVRQA PGKGLEWVSY IRPSGGRTTY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARGG LLLWFRYLKS NYFDYWGQGT 120LVTVSSASTK GPSVFPLAPS SKS 143 X66-F01 LCQDIQMTQSPS SLSAFVGDRV TITCRASQPI DNYLNWYHQK PGKAPKLLIY AASRLQSGVP  60SRLSGSGFGT DFTLTISSLQ PEDFGNYYCQ QSYTVPYTFG GGTKVEIR 108 X66-F01 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS AYSMIWVRQA PGKGLEWVSY IRPSGGRTTY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARGG LLLWFSELKS NYFDYWGQGT 120LVTVSSASTK GPSVFPLAPS SKS 143 X66-H11 LCQDIQMTQSPS SLSAFVGDRV TITCRASQPI DNYLNWYHQK PGKAPKLLIY AASRLQSGVP  60SRLSGSGFGT DFTLTISSLQ PEDFGNYYCQ QSYTVPYTFG GGTKVEIR 108 X66-H11 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS AYSMIWVRQA PGKGLEWVSY IRPSGGRTTY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARGG LLLWFTELKS NYFDYWGQGT 120LVTVSSASTK GPSVFPLAPS SKS 143 X66-C02 LCQDIQMTQSPS SLSAFVGDRV TITCRASQPI DNYLNWYHQK PGKAPKLLIY AASRLQSGVP  60SRLSGSGFGT DFTLTISSLQ PEDFGNYYCQ QSYTVPYTFG GGTKVEIR 108 X66-C02 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS AYSMIWVRQA PGKGLEWVSY IRPSGGRTTY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARGG LLLWFVELKS NYFDYWGQGT 120LVTVSSASTK GPSVFPLAPS SKS 143 X66-F09 LCQDIQMTQSPS SLSAFVGDRV TITCRASQPI DNYLNWYHQK PGKAPKLLIY AASRLQSGVP  60SRLSGSGFGT DFTLTISSLQ PEDFGNYYCQ QSYTVPYTFG GGTKVEIR 108 X66-F09 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS AYSMIWVRQA PGKGLEWVSY IRPSGGRTTY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARGG LLLWFWELKS NYFDYWGQGT 120LVTVSSASTK GPSVFPLAPS SKS 143 X66-G08 LCQDIQMTQSPS SLSAFVGDRV TITCRASQPI DNYLNWYHQK PGKAPKLLIY AASRLQSGVP  60SRLSGSGFGT DFTLTISSLQ PEDFGNYYCQ QSYTVPYTFG GGTKVEIR 108 X66-G08 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS AYSMIWVRQA PGKGLEWVSY IRPSGGRTTY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARGG LLLWFYELKS NYFDYWGQGT 120LVTVSSASTK GPSVFPLAPS SKS 143 X67-C09 LCQDIQMTQSPS SLSAFVGDRV TITCRASQPI DNYLNWYHQK PGKAPKLLIY AASRLQSGVP  60SRLSGSGFGT DFTLTISSLQ PEDFGNYYCQ QSYTVPYTFG GGTKVEIR 108 X67-C09 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS AYSMIWVRQA PGKGLEWVSY IRPSGGRTTY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARGG LLLWGRELKS NYFDYWGQGT 120LVTVSSASTK GPSVFPLAPS SKS 143 X67-B04 LCQDIQMTQSPS SLSAFVGDRV TITCRASQPI DNYLNWYHQK PGKAPKLLIY AASRLQSGVP  60SRLSGSGFGT DFTLTISSLQ PEDFGNYYCQ QSYTVPYTFG GGTKVEIR 108 X67-B04 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS AYSMIWVRQA PGKGLEWVSY IRPSGGRTTY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARGG LLLWKRELKS NYFDYWGQGT 120LVTVSSASTK GPSVFPLAPS SKS 143 X67-G09 LCQDIQMTQSPS SLSAFVGDRV TITCRASQPI DNYLNWYHQK PGKAPKLLIY AASRLQSGVP  60SRLSGSGFGT DFTLTISSLQ PEDFGNYYCQ QSYTVPYTFG GGTKVEIR 108 X67-G09 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS AYSMIWVRQA PGKGLEWVSY IRPSGGRTTY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARGG LLLWLRELKS NYFDYWGQGT 120LVTVSSASTK GPSVFPLAPS SKS 143 X67-C03 LCQDIQMTQSPS SLSAFVGDRV TITCRASQPI DNYLNWYHQK PGKAPKLLIY AASRLQSGVP  60SRLSGSGFGT DFTLTISSLQ PEDFGNYYCQ QSYTVPYTFG GGTKVEIR 108 X67-C03 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS AYSMIWVRQA PGKGLEWVSY IRPSGGRTTY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARGG LLLWMRELKS NYFDYWGQGT 120LVTVSSASTK GPSVFPLAPS SKS 143 X67-D03 LCQDIQMTQSPS SLSAFVGDRV TITCRASQPI DNYLNWYHQK PGKAPKLLIY AASRLQSGVP  60SRLSGSGFGT DFTLTISSLQ PEDFGNYYCQ QSYTVPYTFG GGTKVEIR 108 X67-D03 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS AYSMIWVRQA PGKGLEWVSY IRPSGGRTTY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARGG LLLWNRELKS NYFDYWGQGT 120LVTVSSASTK GPSVFPLAPS SKS 143 X67-B05 LCQDIQMTQSPS SLSAFVGDRV TITCRASQPI DNYLNWYHQK PGKAPKLLIY AASRLQSGVP  60SRLSGSGFGT DFTLTISSLQ PEDFGNYYCQ QSYTVPYTFG GGTKVEIR 108 X67-B05 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS AYSMIWVRQA PGKGLEWVSY IRPSGGRTTY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARGG LLLWPRELKS NYFDYWGQGT 120LVTVSSASTK GPSVFPLAPS SKS 143 X67-F01 LCQDIQMTQSPS SLSAFVGDRV TITCRASQPI DNYLNWYHQK PGKAPKLLIY AASRLQSGVP  60SRLSGSGFGT DFTLTISSLQ PEDFGNYYCQ QSYTVPYTFG GGTKVEIR 108 X67-F01 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS AYSMIWVRQA PGKGLEWVSY IRPSGGRTTY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARGG LLLWQRELKS NYFDYWGQGT 120LVTVSSASTK GPSVFPLAPS SKS 143 X67-G05 LCQDIQMTQSPS SLSAFVGDRV TITCRASQPI DNYLNWYHQK PGKAPKLLIY AASRLQSGVP  60SRLSGSGFGT DFTLTISSLQ PEDFGNYYCQ QSYTVPYTFG GGTKVEIR 108 X67-G05 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS AYSMIWVRQA PGKGLEWVSY IRPSGGRTTY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARGG LLLWRRELKS NYFDYWGQGT 120LVTVSSASTK GPSVFPLAPS SKS 143 X67-B03 LCQDIQMTQSPS SLSAFVGDRV TITCRASQPI DNYLNWYHQK PGKAPKLLIY AASRLQSGVP  60SRLSGSGFGT DFTLTISSLQ PEDFGNYYCQ QSYTVPYTFG GGTKVEIR 108 X67-B03 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS AYSMIWVRQA PGKGLEWVSY IRPSGGRTTY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARGG LLLWSRELKS NYFDYWGQGT 120LVTVSSASTK GPSVFPLAPS SKS 143 X67-F10 LCQDIQMTQSPS SLSAFVGDRV TITCRASQPI DNYLNWYHQK PGKAPKLLIY AASRLQSGVP  60SRLSGSGFGT DFTLTISSLQ PEDFGNYYCQ QSYTVPYTFG GGTKVEIR 108 X67-F10 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS AYSMIWVRQA PGKGLEWVSY IRPSGGRTTY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARGG LLLWTRELKS NYFDYWGQGT 120LVTVSSASTK GPSVFPLAPS SKS 143 X67-H01 LCQDIQMTQSPS SLSAFVGDRV TITCRASQPI DNYLNWYHQK PGKAPKLLIY AASRLQSGVP  60SRLSGSGFGT DFTLTISSLQ PEDFGNYYCQ QSYTVPYTFG GGTKVEIR 108 X67-H01 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS AYSMIWVRQA PGKGLEWVSY IRPSGGRTTY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARGG LLLWWRELKS NYFDYWGQGT 120LVTVSSASTK GPSVFPLAPS SKS 143 X67-F08 LCQDIQMTQSPS SLSAFVGDRV TITCRASQPI DNYLNWYHQK PGKAPKLLIY AASRLQSGVP  60SRLSGSGFGT DFTLTISSLQ PEDFGNYYCQ QSYTVPYTFG GGTKVEIR 108 X67-F08 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS AYSMIWVRQA PGKGLEWVSY IRPSGGRTTY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARGG LLLWYRELKS NYFDYWGQGT 120LVTVSSASTK GPSVFPLAPS SKS 143

TABLE 6CDR Amino Acid Sequences of Optimized Antibody Inhibitor of pKal Based on M142-H08 (SEQ ID NOs: 1086-1092) Ki,   app LV-CDR2 HV-CDR1 (nM)LV-CDR1 (SEQ ID LV-CDR3 (SEQ ID HV-CDR3^(a) Initial of (SEQ ID NO:(SEQ ID NO: HV-CDR2 (SEQ ID NOs: Name IgG NO: 1086) 1087) NO: 1088)1089) (SEQ ID NO: 1090) 1091 and 1092) X67-D03 0.1 RASQPIDNYLN AASRLQSQQSYTVPYT AYSMI YIRPSGGRTTYADSVKG GGLLLWNRELKSNYFDY X67-G04 0.35RASQPIDNYLN AASRLQS QQSYTVPYT AYSMI YIRPSGGRTTYADSVKG GGLLLWARELKSNYFDY^(a)The F to N substitution (in bold) in the CDR3 of the M142-H08 givesX67-D03, an IgG that is not cleaved during expression and is a potentinhibitor of human. Similarly, the F to A substitution gives X67-G04,which is also not cleaved.

TABLE 7CDR Amino Acid Sequences of Affinity Matured Antibody Inhibitors of pKal  Discovered using ROLIC (SEQ ID NOs: 1093-1113) HV- LV-CDR2 LV-CDR3 CDR1LV-CDR1 (SEQ ID (SEQ ID (SEQ Ki, (SEQ ID NOs: NOs: ID HV-CDR2 HV-CDR3Initial app NOs: 1099- 1105- NO: (SEQ ID (SEQ ID Name (nM) 1093-1098)1104) 1110) 1111) NO: 1112) NO: 1113) X59-C07 6.1 RAGRSISTYVN AASSLQSQQSQSTPYT HYLMT YISPSGGHTIYADSVKG VARGIAARSRTSYFDY X60-D01 2.0RASQIVSSRYLA GAASRAT QQTYSSPFT HYLMT YISPSGGHTIYADSVKG VARGIAARSRTSYFDYX63-G10 9.0 RASQSISNYLN AASSLQS QQSYTSPYT HYLMT YISPSGGHTIYADSVKGVARGIAARSRTSYFDY X64-F04 1.9 RASQIVSSNYLA GASNRAT QQSFNIPYT HYLMTYISPSGGHTIYADSVKG VARGIAARSRTSYFDY X63-G06 0.4 RTSQFVNSNYLA GASSRATQQSSRTPWT HYLMT YISPSGGHTIYADSVKG VARGIAARSRTSYFDY (Fab) X81-B01^(a) 0.2RTSQFVNSNYLA GASSRAT QQSSRTPWT HYLMT YISPSGGHTIYADSVKG VARGIAARSRTSYFDY(IgG) ^(a)X81-B01 is the codon optimized and germlined version ofX63-G06 as a full length human IgG produced in HEK 293T cells.

Amino acid sequences of light chain (LC) and heavy chain (HC) variabledomain of affinity matured antibody inhibitors of pKal discovered usingROLIC are shown below (SEQ ID NOs:1114-1123).

X59-C07 LCQDIQMTQSPS SLSASVGDRV TVTCRAGRSI STYVNWYQQK PGKAPKLLIY AASSLQSGVP  60SRFSGSRSGT DFTLTISSLQ PEDFATYYCQ QSQSTPYTFG QGTKLEVK 108 X59-C07 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS HYLMTWVRQA PGKGLEWVSY ISPSGGHTIY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARVA RGIAARSRTS YFDYWGQGTL 120VTVSSASTKG PSVFPLAPSS KS 142 X60-D01 LCQDIQMTQSPG TLSLSPGERA TLSCRASQIV SSRYLAWYQQ RPGQAPRLLI YGAASRATGI  60PDRFSGSGSG TDFTLTISSL QAEDFATYYC QQTYSSPFTF GQGTKMEIK 109 X60-D01 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS HYLMTWVRQA PGKGLEWVSY ISPSGGHTIY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARVA RGIAARSRTS YFDYWGQGTL 120VTVSSASTKG PSVFPLAPSS KS 142 X63-G06 LCQDIQMTQSPG TLSLSPGERA TLSCRTSQFV NSNYLAWYQQ TPGQAPRLLI YGASSRATGI  60PDRFSGTGYG TDFTLTISRL EPEDYGTYYC QQSSRTPWTF GQGTRVEIK 109 X63-G06 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS HYLMTWVRQA PGKGLEWVSY ISPSGGHTIY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARVA RGIAARSRTS YFDYWGQGTL 120VTVSSASTKG PSVFPLAPSS KS 142 X63-G10 LCQDIQMTQSPD SLSASVGDRV TITCRASQSI SNYLNWYQQK PGKAPKLLIY AASSLQSGVP  60SRFSGSGSGT DFTLTISGLQ PEDFASYYCQ QSYTSPYTFV QGTKLEIKRT 110 X63-G10 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS HYLMTWVRQA PGKGLEWVSY ISPSGGHTIY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARVA RGIAARSRTS YFDYWGQGTL 120VTVSSASTKG PSVFPLAPSS KS 142 X64-F04 LCQDIQMTQSPA TLSLSPGERA TLSCRASQIV SSNYLAWYQQ KPGQAPRLLI YGASNRATGI  60PDRFSGSGSG TEFTLTISSL QSEDFAIYYC QQSFNIPYTF GQGTRVDIK 109 X64-F04 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS HYLMTWVRQA PGKGLEWVSY ISPSGGHTIY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARVA RGIAARSRTS YFDYWGQGTL 120VTVSSASTKG PSVFPLAPSS KS 142

X81-B01 is the germlined IgG produced in HEK 293T cells version of theX63-G06 Fab, as indicated above.

X101-A01 (aka DX-2922) is the germlined IgG produced in CHO cellsversion of the X63-G06 Fab

Example 4: Affinity Maturation

In addition to optimizing the sequence of the clipped antibody(M142-H08), we also performed affinity maturation on two of theantibodies identified by phage display (M162-A04 and M160-G12). Both ofthese antibodies inhibit human pKal with single digit nanomolar potency,appear specific to pKal, and do not bind prekallikrein (Table 3). Wefirst performed a novel form of light chain shuffling called ROLIC(Rapid Optimization of Light Chains) on M162-A04 and M160-G12 (see,e.g., WO 2009/102927 and U.S. 2009-0215119). From the screening of theantibodies discovered by ROLIC we identified one antibody withsubnamolar potency (X63-G06) that shared the same heavy chain asM160-G12. We then constructed HV-CDR3 spiking affinity maturationlibraries based on CDR3 sequences in M162-A04 and X63-G06 (describedbelow).

Affinity Maturation by ROLIC.

We used ROLIC to affinity mature the two leads from Table 3 that werenot cleaved (M162-A04 and M160-G12). This process identified oneantibody that inhibits pKal with a subnanomolar K_(i,app) (Table 7).X63-G06 inhibits pKal with a K_(i,app) of approximately 0.4 nM as a Fabfragment. When this antibody was converted to an IgG that is germlinedand sequenced optimized for CHO cell expression (X81-B01) it was foundto inhibit pKal with a K_(i,app) of approximately 0.2 nM.

Example 5: Affinity Maturation of Heavy Chain CDR1/2 and CDR3

We used two additional affinity maturation strategies to identify highlypotent antibodies based on two different parental antibody inhibitorleads: M162-A04 and X63-G06. One approach was to generate libraries thatshuffled the CDR1/2 of the HC of two different parental antibodyinhibitor leads (M162-A04 and X63-G06) against additional CDR1/2diversity. Another approach was to create heavy chain CDR3 spikinglibraries based on these leads.

The 82 antibodies that were discovered based on improvements in M162-A04due to modifications in either the CDR1/2 or CDR3 region are shown inTable 8. Inhibition screening with 10 nM antibody (as Fab fragments)revealed that there were 33 antibodies that inhibited pKal activity byover 90%. Several antibodies were shown to be subnanomolar inhibitors ofhuman pKal.

The 62 antibodies that were discovered based on improvements in X63-G06due to modifications in either the CDR1/2 or CDR3 region are shown inTable 9. Inhibition screening with 10 nM antibody (as Fab fragments)revealed that there were 24 antibodies that inhibited pKal activity byover 90%. Several antibodies were shown to be subnanomolar inhibitors ofhuman pKal.

TABLE 8Sequences of Antibodies Obtained from CDR1/2 and CDR3 Spiking Affinity Maturation Libraries Based on M162-A04 (SEQ ID NOs: 1124-1372) HV- CDR1% in human (SEQ hibi- pKal LV-CDR2 LV-CDR3 ID HV-CDR3 tion Ki, LV-CDR1(SEQ ID (SEQ ID NOs: HV-CDR2 (SEQ ID Antibody at 10 app (SEQ ID NO: NO:1127- (SEQ ID NOs: NOs: I.D. nM (nM) NO: 1124) 1125) 1126) 1208)1209-1290) 1291-1372) M202-A12 97.5 0.2 RASQSISSWLA KASTLES QQYNTYWTHYIMM GIYSSGGITVYADSVKG QRTGVPRRDSFNI M196-C06 97.2 0.1 RASQSISSWLAKASTLES QQYNTYWT IYSMH SIYPSRGMTWYADSVKG RRTGIPRRDAFDI M198-F09 96.9 0.2RASQSISSWLA KASTLES QQYNTYWT VYNMH SIYPSGGMTYYADSVKG RRTGIPRRDAFDIM199-A08 96.4 0.06 RASQSISSWLA KASTLES QQYNTYWT HYIMM GIYSSGGITVYADSVKGRRIGVPRRDEFDI M202-C01 96.3 0.1 RASQSISSWLA KASTLES QQYNTYWT HYIMMGIYSSGGITVYADSVKG RRTGVPRWDDFDI M198-A06 96.1 0.4 RASQSISSWLA KASTLESQQYNTYWT IYSMH SIYSSGGPTKYADSVKG RRTGIPRRDAFDI M200-D03 95.9 0.1RASQSISSWLA KASTLES QQYNTYWT HYIMM GIYSSGGITVYADSVKG RRIGVPRRDSFDMM202-H03 95.7 0.1 RASQSISSWLA KASTLES QQYNTYWT HYIMM GIYSSGGITVYADSVKGRRTGVPRWDDFDI M201-A07 95.7 0.1 RASQSISSWLA KASTLES QQYNTYWT HYIMMGIYSSGGITVYADSVKG RRTGVPRRDEFDI M197-A01 95.3 RASQSISSWLA KASTLESQQYNTYWT IYDMI SIYPSGGNTSYADSVKG RRTGIPRRDAFDI M202-D09 95.0 0.4RASQSISSWLA KASTLES QQYNTYWT HYIMM GIYSSGGITVYADSVKG RRIGVPRRDSFDIM197-A09 94.9 0.6 RASQSISSWLA KASTLES QQYNTYWT VYNMH SIYPSGGMTTYADSVKGRRTGIPRRDAFDI M198-G07 94.9 RASQSISSWLA KASTLES QQYNTYWT IYDMTSIYPSGGQTIYADSVKG RRTGIPRRDAFDI M200-A10 94.3 0.3 RASQSISSWLA KASTLESQQYNTYWT HYIMM GIYSSGGITVYADSVKG RRTGVPRRDSFDI M197-H10 94.1 RASQSISSWLAKASTLES QQYNTYWT SYNMH SIVPSGGKTNYADSVKG RRTGIPRRDAFDI M196-D12 94.1 0.2RASQSISSWLA KASTLES QQYNTYWT RYSMR VIYPSGGQTYYADSVKG RRTGIPRRDAFDIM197-A08 93.7 RASQSISSWLA KASTLES QQYNTYWT IYSMQ SIGSSGGKTLYADSVKGRRTGIPRRDAFDI M198-B09 93.5 RASQSISSWLA KASTLES QQYNTYWT VYSMTSIGSSGGSTTYADSVKG RRTGIPRRDAFDI M198-E09 93.1 RASQSISSWLA KASTLESQQYNTYWT IYDMN SIYPSGGRTRYADSVKG RRTGIPRRDAFDI M202-B03 93.1 0.3RASQSISSWLA KASTLES QQYNTYWT HYIMM GIYSSGGITVYADSVKG RRTGVPRRDDFDIM198-C10 93.0 RASQSISSWLA KASTLES QQYNTYWT HYMGMN SIVPSGGWTQYADSVKGRRTGIPRRDAFDI M197-E12 93.0 RASQSISSWLA KASTLES QQYNTYWT TYTMRSIYPSGGKTQYADSVKG RRTGIPRRDAFDI M198-F04 92.9 RASQSISSWLA KASTLESQQYNTYWT IYDMW SIRPSGGITKYADSVKG RRTGIPRRDAFDI M197-H11 92.9 RASQSISSWLAKASTLES QQYNTYWT IYNMI SIYPSGGWTTYADSVKG RRTGIPRRDAFDI M197-F01 92.6RASQSISSWLA KASTLES QQYNTYWT IYHMY SIGPSGGPTGYADSVKG RRTGIPRRDAFDIM198-E11 92.5 RASQSISSWLA KASTLES QQYNTYWT TYSMY SIYPSGGLTWYADSVKGRRTGIPRRDAFDI M202-C09 92.3 0.3 RASQSISSWLA KASTLES QQYNTYWT HYIMMGIYSSGGITVYADSVKG RRIGVPRRDDFDI M198-H08 92.3 RASQSISSWLA KASTLESQQYNTYWT IYDMY SIGPSGGPTAYADSVKG RRTGIPRRDAFDI M198-F08 91.8 RASQSISSWLAKASTLES QQYNTYWT VYSMW SISSSGGMTEYADSVKG RRTGIPRRDAFDI M202-E06 91.5RASQSISSWLA KASTLES QQYNTYWT HYIMM GIYSSGGITVYADSVKG RRRGVPRRDDFDIM195-D12 90.8 RASQSISSWLA KASTLES QQYNTYWT IYGMF GIGPSGGPTKYADSVKGRRTGIPRRDAFDI M197-F03 90.7 RASQSISSWLA KASTLES QQYNTYWT IYSMFSIGPSGGVTHYADSVKG RRTGIPRRDAFDI M198-E02 90.3 RASQSISSWLA KASTLESQQYNTYWT IYSMY YIRPSGGNTKYADSVKG RRTGIPRRDAFDI M198-A02 89.1 RASQSISSWLAKASTLES QQYNTYWT RYSMI SIWSSGGATEYADSVKG RRTGIPRRDAFDI M202-A01 88.9RASQSISSWLA KASTLES QQYNTYWT HYIMM GIYSSGGITVYADSVKG RRIGVPRRDAFDIM202-G03 88.3 RASQSISSWLA KASTLES QQYNTYWT HYIMM GIYSSGGITVYADSVKGRRTGVPRRDSFEI M195-B12 87.7 RASQSISSWLA KASTLES QQYNTYWT KYWMYYIRPSGGQTYYADSVKG RRTGIPRRDAFDI M198-A07 86.1 RASQSISSWLA KASTLESQQYNTYWT RYQMH WISPSGGITGYADSVKG RRTGIPRRDAFDI M198-H02 85.8 RASQSISSWLAKASTLES QQYNTYWT PYNMY WIVPGGVTKYADSVKG RRTGIPRRDAFDI M200-H07 85.4RASQSISSWLA KASTLES QQYNTYWT HYIMM GIYSSGGITVYADSVKG RRTGVPRRNAFDNM201-H06 84.6 RASQSISSWLA KASTLES QQYNTYWT HYIMM GIYSSGGITVYADSVKGRRTGVPRRDAFDI M202-F06 84.2 RASQSISSWLA KASTLES QQYNTYWT HYIMMGIYSSGGITVYADSVKG RRTGVPRWDAFDI M195-C12 84.2 RASQSISSWLA KASTLESQQYNTYWT MYQMF SISPGGGTQYADSVKG RRTGIPRRDAFDI M202-H05 84.0 RASQSISSWLAKASTLES QQYNTYWT HYIMM GIYSSGGITVYADSVKG RRTGVPRRDVFDI M198-C05 83.9RASQSISSWLA KASTLES QQYNTYWT RYKMY VIGPSGGATFYADSVKG RRTGIPRRDAFDIM196-H03 83.9 RASQSISSWLA KASTLES QQYNTYWT RYVMW SISPSGDTHYADSVKGRRTGIPRRDAFDI M200-E11 83.2 RASQSISSWLA KASTLES QQYNTYWT HYIMMGIYSSGGITVYADSVKG RRTGVPRRDAFDN M202-B04 81.9 RASQSISSWLA KASTLESQQYNTYWT HYIMM GIYSSGGITVYADSVKG RRSGVPRRDDFDI M202-A04 81.2 RASQSISSWLAKASTLES QQYNTYWT HYIMM GIYSSGGITVYADSVKG RRKGIPRRDDFDI M198-B12 80.7RASQSISSWLA KASTLES QQYNTYWT KYSMA GIYPSGGRTLYADSVKG RRTGIPRRDAFDIM198-A09 77.3 RASQSISSWLA KASTLES QQYNTYWT IYFMS SIRSSGGPTWYADSVKGRRTGIPRRDAFDI M198-C06 76.5 RASQSISSWLA KASTLES QQYNTYWT QYFMHYIYPSGGMTEYADSVKG RRTGIPRRDAFDI M198-C09 75.4 RASQSISSWLA KASTLESQQYNTYWT IYTMY SISPSGGWTYYADSVKG RRTGIPRRDAFDI M195-B02 75.1 RASQSISSWLAKASTLES QQYNTYWT PYLMW YIGPSGGPTHYADSVKG RRTGIPRRDAFDI M198-F12 74.6RASQSISSWLA KASTLES QQYNTYWT IYTMM SIWSSGGQTKYADSVKG RRTGIPRRDAFDIM201-H08 74.5 RASQSISSWLA KASTLES QQYNTYWT HYIMM GIYSSGGITVYADSVKGRRTGVPRRDALDN M202-C02 74.3 RASQSISSWLA KASTLES QQYNTYWT HYIMMGIYSSGGITVYADSVKG RRPGVPRRDAFDI M198-C03 72.4 RASQSISSWLA KASTLESQQYNTYWT RYSMS GISPSGGETSYADSVKG RRTGIPRRDAFDI M198-A08 72.3 RASQSISSWLAKASTLES QQYNTYWT WYMMQ RISPSGGTTYADSVKG RRTGIPRRDAFDI M195-A02 71.3RASQSISSWLA KASTLES QQYNTYWT QYMMM GISSSGGHTDYADSVKG RRTGIPRRDAFDIM197-G10 67.6 RASQSISSWLA KASTLES QQYNTYWT VYAMR SIYPSGGKTWYADSVKGRRTGIPRRDAFDI M195-G02 67.5 RASQSISSWLA KASTLES QQYNTYWT PYNMMSIWPSGGTTDYADSVKG RRTGIPRRDAFDI M196-D02 66.2 RASQSISSWLA KASTLESQQYNTYWT VYSMH VIGPSGGITLYADSVKG RRTGIPRRDAFDI M199-A11 65.4 RASQSISSWLAKASTLES QQYNTYWT HYIMM GIYSSGGITVYADSVKG RRRGIPRRDAFDI M200-F01 65.1RASQSISSWLA KASTLES QQYNTYWT HYIMM GIYSSGGITVYADSVKG RRMGIPRRNAFDIM198-D12 63.5 0.7 RASQSISSWLA KASTLES QQYNTYWT LYVMY YIVPSGGPTAYADSVKGRRTGIPRRDAFDI M197-C12 56.4 RASQSISSWLA KASTLES QQYNTYWT PYDMLYIVSSGGLTKYADSVKG RRTGIPRRDAFDI M198-G03 53.8 RASQSISSWLA KASTLESQQYNTYWT QYTMV WIYSSRANYADSVKG RRTGIPRRDAFDI M199-B01 53.4 RASQSISSWLAKASTLES QQYNTYWT HYIMM GIYSSGGITVYADSVKG RRTGIPRRDAFDN M202-A08 52.9RASQSISSWLA KASTLES QQYNTYWT HYIMM GIYSSGGITVYADSVKG RRTGIPRWDAFDIM195-A12 51.7 RASQSISSWLA KASTLES QQYNTYWT PYMMM GIYPSGGYTVYADSVKGRRTGIPRRDAFDI M202-E03 51.4 RASQSISSWLA KASTLES QQYNTYWT HYIMMGIYSSGGITVYADSVKG RRTGIPRRDAFEI M196-G12 51.1 RASQSISSWLA KASTLESQQYNTYWT NYSMD RIYSSGGGTIYADSVKG RRTGIPRRDAFDI M195-F12 45.5 RASQSISSWLAKASTLES QQYNTYWT HYVMM YIVPSGGVTAYADSVKG RRTGIPRRDAFDI M200-B01 42.6RASQSISSWLA KASTLES QQYNTYWT HYIMM GIYSSGGITVYADSVKG RRTGIPRRDAFDSM198-H09 41.1 RASQSISSWLA KASTLES QQYNTYWT IYLMI YIGPSGGPTEYADSVKGRRTGIPRRDAFDI M195-E12 38.0 RASQSISSWLA KASTLES QQYNTYWT YYIMFYISPSGGYTHYADSVKG RRTGIPRRDAFDI M201-A06 36.8 RASQSISSWLA KASTLESQQYNTYWT HYIMM GIYSSGGITVYADSVKG RRTGIPRRDVFDI M202-A10 36.3 RASQSISSWLAKASTLES QQYNTYWT HYIMM GIYSSGGITVYADSVKG RRTGIPRRDSFDI M197-G11 19.2RASQSISSWLA KASTLES QQYNTYWT TYAMV SIYPSGGITTYADSVKG RRTGIPRRDAFDIM201-F11 15.7 RASQSISSWLA KASTLES QQYNTYWT HYIMM GIYSSGGITVYADSVKGRRSGIPRRDAFDI M198-A01 13.8 RASQSISSWLA KASTLES QQYNTYWT PYTMISISSSGGMTPYADSVKG RRTGIPRRDAFDI

Amino acid sequences of light chain (LC) and heavy chain (HC) variabledomain of pKal antibodies obtained from CDR1/2 and CDR3 spiking affinitymaturation libraries based on M162-A04 (SEQ ID NOs:1373-1536).

M195-A02 LCQDIQMTQSPS TLSASVGDRV TITCRASQSI SSWLAWYQQK PGKAPNLLIY KASTLESGVP  60SRFSGSGSGT EFTLTISSLQ PDDFATYYCQ QYNTYWTFGQ GTKVEIK 107 M195-A02 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS QYMMMWVRQA PGKGLEWVSG ISSSGGHTDY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAYRR TGIPRRDAFD IWGQGTMVTV 120SSASTKGPSV FPLAPSSKS 139 M195-A12 LCQDIQMTQSPS TLSASVGDRV TITCRASQSI SSWLAWYQQK PGKAPNLLIY KASTLESGVP  60SRFSGSGSGT EFTLTISSLQ PDDFATYYCQ QYNTYWTFGQ GTKVEIK 107 M195-A12 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS PYMMMWVRQA PGKGLEWVSG IYPSGGYTVY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAYRR TGIPRRDAFD IWGQGTMVTV 120SSASTKGPSV FPLAPSSKS 139 M195-B02 LCQDIQMTQSPS TLSASVGDRV TITCRASQSI SSWLAWYQQK PGKAPNLLIY KASTLESGVP  60SRFSGSGSGT EFTLTISSLQ PDDFATYYCQ QYNTYWTFGQ GTKVEIK 107 M195-B02 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS PYLMWWVRQA PGKGLEWVSY IGPSGGPTHY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAYRR TGIPRRDAFD IWGQGTMVTV 120SSASTKGPSV FPLAPSSKS 139 M195-B12 LCQDIQMTQSPS TLSASVGDRV TITCRASQSI SSWLAWYQQK PGKAPNLLIY KASTLESGVP  60SRFSGSGSGT EFTLTISSLQ PDDFATYYCQ QYNTYWTFGQ GTKVEIK 107 M195-B12 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS KYWMYWVRQA PGKGLEWVSY IRPSGGQTYY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAYRR TGIPRRDAFD IWGQGTMVTV 120SSASTKGPSV FPLAPSSKS 139 M195-C12 LCQDIQMTQSPS TLSASVGDRV TITCRASQSI SSWLAWYQQK PGKAPNLLIY KASTLESGVP  60SRFSGSGSGT EFTLTISSLQ PDDFATYYCQ QYNTYWTFGQ GTKVEIK 107 M195-C12 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS MYQMFWVRQA PGKGLEWVSS ISPGGGTQYA  60DSVKGRFTIS RDNSKNTLYL QMNSLRAEDT AVYYCAYRRT GIPRRDAFDI WGQGTMVTVS 120SASTKGPSVF PLAPSSKS 138 M195-D12 LCQDIQMTQSPS TLSASVGDRV TITCRASQSI SSWLAWYQQK PGKAPNLLIY KASTLESGVP  60SRFSGSGSGT EFTLTISSLQ PDDFATYYCQ QYNTYWTFGQ GTKVEIK 107 M195-D12 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS IYGMFWVRQA PGKGLEWVSG IGPSGGPTKY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAYRR TGIPRRDAFD IWGQGTMVTV 120SSASTKGPSV FPLAPSSKS 139 M195-E12 LCQDIQMTQSPS TLSASVGDRV TITCRASQSI SSWLAWYQQK PGKAPNLLIY KASTLESGVP  60SRFSGSGSGT EFTLTISSLQ PDDFATYYCQ QYNTYWTFGQ GTKVEIK 107 M195-E12 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS YYIMFWVRQA PGKGLEWVSY ISPSGGYTHY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAYRR TGIPRRDAFD IWGQGTMVTV 120SSASTKGPSV FPLAPSSKS 139 M195-F12 LCQDIQMTQSPS TLSASVGDRV TITCRASQSI SSWLAWYQQK PGKAPNLLIY KASTLESGVP  60SRFSGSGSGT EFTLTISSLQ PDDFATYYCQ QYNTYWTFGQ GTKVEIK 107 M195-F12 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS HYVMMWVRQA PGKGLEWVSY IVPSGGVTAY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAYRR TGIPRRDAFD IWGQGTMVTV 120SSASTKGPSV FPLAPSSKS 139 M0195-G02 LCQDIQMTQSPS TLSASVGDRV TITCRASQSI SSWLAWYQQK PGKAPNLLIY KASTLESGVP  60SRFSGSGSGT EFTLTISSLQ PDDFATYYCQ QYNTYWTFGQ GTKVEIK 107 M195-G02 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS PYNMMWVRQA PGKGLEWVSS IWPSGGTTDY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAYRR TGIPRRDAFD IWGQGTMVTV 120SSASTKGPSV FPLAPSSKS 139 M196-C06 LCQDIQMTQSPS TLSASVGDRV TITCRASQSI SSWLAWYQQK PGKAPNLLIY KASTLESGVP  60SRFSGSGSGT EFTLTISSLQ PDDFATYYCQ QYNTYWTFGQ GTKVEIK 107 M196-C06 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS IYSMHWVRQA PGKGLEWVSS IYPSRGMTWY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAYRR TGIPRRDAFD IWGQGTMVTV 120SSASTKGPSV FPLAPSSKS 139 M196-D02 LCQDIQMTQSPS TLSASVGDRV TITCRASQSI SSWLAWYQQK PGKAPNLLIY KASTLESGVP  60SRFSGSGSGT EFTLTISSLQ PDDFATYYCQ QYNTYWTFGQ GTKVEIK 107 M196-D02 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS VYSMHWVRQA PGKGLEWVSV IGPSGGITLY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAYRR TGIPRRDAFD IWGQGTMVTV 120SSASTKGPSV FPLAPSSKS 139 M196-D12 LCQDIQMTQSPS TLSASVGDRV TITCRASQSI SSWLAWYQQK PGKAPNLLIY KASTLESGVP  60SRFSGSGSGT EFTLTISSLQ PDDFATYYCQ QYNTYWTFGQ GTKVEIK 107 M196-D12 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS RYSMRWVRQA PGKGLEWVSV IYPSGGQTYY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAYRR TGIPRRDAFD IWGQGTMVTV 120SSASTKGPSV FPLAPSSKS 139 M196-G12 LCQDIQMTQSPS TLSASVGDRV TITCRASQSI SSWLAWYQQK PGKAPNLLIY KASTLESGVP  60SRFSGSGSGT EFTLTISSLQ PDDFATYYCQ QYNTYWTFGQ GTKVEIK 107 M196-G12 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS NYSMDWVRQA PGKGLEWVSR IYSSGGGTIY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAYRR TGIPRRDAFD IWGQGTMVTV 120SSASTKGPSV FPLAPSSKS 139 M196-H03 LCQDIQMTQSPS TLSASVGDRV TITCRASQSI SSWLAWYQQK PGKAPNLLIY KASTLESGVP  60SRFSGSGSGT EFTLTISSLQ PDDFATYYCQ QYNTYWTFGQ GTKVEIK 107 M196-H03 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS RYVMWWVRQA PGKGLEWVSS ISPSGDTHYA  60DSVKGRFTIS RDNSKNTLYL QMNSLRAEDT AVYYCAYRRT GIPRRDAFDI WGQGTMVTVS 120SASTKGPSVF PLAPSSKS 138 M197-A01 LCQDIQMTQSPS TLSASVGDRV TITCRASQSI SSWLAWYQQK PGKAPNLLIY KASTLESGVP  60SRFSGSGSGT EFTLTISSLQ PDDFATYYCQ QYNTYWTFGQ GTKVEIK 107 M197-A01 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS IYDMIWVRQA PGKGLEWVSS IYPSGGNTSY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAYRR TGIPRRDAFD IWGQGTMVTV 120SSASTKGPSV FPLAPSSKS 139 M197-A08 LCQDIQMTQSPS TLSASVGDRV TITCRASQSI SSWLAWYQQK PGKAPNLLIY KASTLESGVP  60SRFSGSGSGT EFTLTISSLQ PDDFATYYCQ QYNTYWTFGQ GTKVEIK 107 M197-A08 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS IYSMQWVRQA PGKGLEWVSS IGSSGGKTLY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAYRR TGIPRRDAFD IWGQGTMVTV 120SSASTKGPSV FPLAPSSKS 139 M197-A09 LCQDIQMTQSPS TLSASVGDRV TITCRASQSI SSWLAWYQQK PGKAPNLLIY KASTLESGVP  60SRFSGSGSGT EFTLTISSLQ PDDFATYYCQ QYNTYWTFGQ GTKVEIK 107 M197-A09 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS VYNMHWVRQA PGKGLEWVSS IYPSGGMTTY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAYRR TGIPRRDAFD IWGQGTMVTV 120SSASTKGPSV FPLAPSSKS 139 M197-C12 LCQDIQMTQSPS TLSASVGDRV TITCRASQSI SSWLAWYQQK PGKAPNLLIY KASTLESGVP  60SRFSGSGSGT EFTLTISSLQ PDDFATYYCQ QYNTYWTFGQ GTKVEIK 107 M197-C12 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS PYDMLWVRQA PGKGLEWVSY IVSSGGLTKY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAYRR TGIPRRDAFD IWGQGTMVTV 120SSASTKGPSV FPLAPSSKS 139 M197-E12 LCQDIQMTQSPS TLSASVGDRV TITCRASQSI SSWLAWYQQK PGKAPNLLIY KASTLESGVP  60SRFSGSGSGT EFTLTISSLQ PDDFATYYCQ QYNTYWTFGQ GTKVEIK 107 M197-E12 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS TYTMRWVRQA PGKGLEWVSS IYPSGGKTQY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAYRR TGIPRRDAFD IWGQGTMVTV 120SSASTKGPSV FPLAPSSKS 139 M197-F01 LCQDIQMTQSPS TLSASVGDRV TITCRASQSI SSWLAWYQQK PGKAPNLLIY KASTLESGVP  60SRFSGSGSGT EFTLTISSLQ PDDFATYYCQ QYNTYWTFGQ GTKVEIK 107 M197-F01 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS IYHMYWVRQA PGKGLEWVSS IGPSGGPTGY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAYRR TGIPRRDAFD IWGQGTMVTV 120SSASTKGPSV FPLAPSSKS 139 M197-F03 LCQDIQMTQSPS TLSASVGDRV TITCRASQSI SSWLAWYQQK PGKAPNLLIY KASTLESGVP  60SRFSGSGSGT EFTLTISSLQ PDDFATYYCQ QYNTYWTFGQ GTKVEIK 107 M197-F03 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS IYSMFWVRQA PGKGLEWVSS IGPSGGVTHY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAYRR TGIPRRDAFD IWGQGTMVTV 120SSASTKGPSV FPLAPSSKS 139 M197-G10 LCQDIQMTQSPS TLSASVGDRV TITCRASQSI SSWLAWYQQK PGKAPNLLIY KASTLESGVP  60SRFSGSGSGT EFTLTISSLQ PDDFATYYCQ QYNTYWTFGQ GTKVEIK 107 M197-G10 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS VYAMRWVRQA PGKGLEWVSS IYPSGGKTWY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAYRR TGIPRRDAFD IWGQGTMVTV 120SSASTKGPSV FPLAPSSKS 139 M197-G11 LCQDIQMTQSPS TLSASVGDRV TITCRASQSI SSWLAWYQQK PGKAPNLLIY KASTLESGVP  60SRFSGSGSGT EFTLTISSLQ PDDFATYYCQ QYNTYWTFGQ GTKVEIK 107 M197-G11 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS TYAMVWVRQA PGKGLEWVSS IYPSGGITTY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAYRR TGIPRRDAFD IWGQGTMVTV 120SSASTKGPSV FPLAPSSKS 139 M197-H10 LCQDIQMTQSPS TLSASVGDRV TITCRASQSI SSWLAWYQQK PGKAPNLLIY KASTLESGVP  60SRFSGSGSGT EFTLTISSLQ PDDFATYYCQ QYNTYWTFGQ GTKVEIK 107 M197-H10 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS SYNMHWVRQA PGKGLEWVSS IVPSGGKTNY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAYRR TGIPRRDAFD IWGQGTMVTV 120SSASTKGPSV FPLAPSSKS 139 M197-H11 LCQDIQMTQSPS TLSASVGDRV TITCRASQSI SSWLAWYQQK PGKAPNLLIY KASTLESGVP  60SRFSGSGSGT EFTLTISSLQ PDDFATYYCQ QYNTYWTFGQ GTKVEIK 107 M197-H11 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS IYNMIWVRQA PGKGLEWVSS IYPSGGWTTY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAYRR TGIPRRDAFD IWGQGTMVTV 120SSASTKGPSV FPLAPSSKS 139 M198-A01 LCQDIQMTQSPS TLSASVGDRV TITCRASQSI SSWLAWYQQK PGKAPNLLIY KASTLESGVP  60SRFSGSGSGT EFTLTISSLQ PDDFATYYCQ QYNTYWTFGQ GTKVEIK 107 M198-A01 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS PYTMIWVRQA PGKGLEWVSS ISSSGGMTPY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAYRR TGIPRRDAFD IWGQGTMVTV 120SSASTKGPSV FPLAPSSKS 139 M198-A02 LCQDIQMTQSPS TLSASVGDRV TITCRASQSI SSWLAWYQQK PGKAPNLLIY KASTLESGVP  60SRFSGSGSGT EFTLTISSLQ PDDFATYYCQ QYNTYWTFGQ GTKVEIK 107 M198-A02 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS RYSMIWVRQA PGKGLEWVSS IWSSGGATEY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAYRR TGIPRRDAFD IWGQGTMVTV 120SSASTKGPSV FPLAPSSKS 139 M198-A06 LCQDIQMTQSPS TLSASVGDRV TITCRASQSI SSWLAWYQQK PGKAPNLLIY KASTLESGVP  60SRFSGSGSGT EFTLTISSLQ PDDFATYYCQ QYNTYWTFGQ GTKVEIK 107 M198-A06 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS IYSMHWVRQA PGKGLEWVSS IYSSGGPTKY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAYRR TGIPRRDAFD IWGQGTMVTV 120SSASTKGPSV FPLAPSSKS 139 M198-A07 LCQDIQMTQSPS TLSASVGDRV TITCRASQSI SSWLAWYQQK PGKAPNLLIY KASTLESGVP  60SRFSGSGSGT EFTLTISSLQ PDDFATYYCQ QYNTYWTFGQ GTKVEIK 107 M198-A07 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS RYQMHWVRQA PGKGLEWVSW ISPSGGITGY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAYRR TGIPRRDAFD IWGQGTMVTV 120SSASTKGPSV FPLAPSSKS 139 M198-A08 LCQDIQMTQSPS TLSASVGDRV TITCRASQSI SSWLAWYQQK PGKAPNLLIY KASTLESGVP  60SRFSGSGSGT EFTLTISSLQ PDDFATYYCQ QYNTYWTFGQ GTKVEIK 107 M198-A08 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS WYMMQWVRQA PGKGLEWVSR ISPSGGTTYA  60DSVKGRFTIS RDNSKNTLYL QMNSLRAEDT AVYYCAYRRT GIPRRDAFDI WGQGTMVTVS 120SASTKGPSVF PLAPSSKS 138 M198-A09 LCQDIQMTQSPS TLSASVGDRV TITCRASQSI SSWLAWYQQK PGKAPNLLIY KASTLESGVP  60SRFSGSGSGT EFTLTISSLQ PDDFATYYCQ QYNTYWTFGQ GTKVEIK 107 M198-A09 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS IYFMSWVRQA PGKGLEWVSS IRSSGGPTWY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAYRR TGIPRRDAFD IWGQGTMVTV 120SSASTKGPSV FPLAPSSKS 139 M198-B09 LCQDIQMTQSPS TLSASVGDRV TITCRASQSI SSWLAWYQQK PGKAPNLLIY KASTLESGVP  60SRFSGSGSGT EFTLTISSLQ PDDFATYYCQ QYNTYWTFGQ GTKVEIK 107 M198-B09 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS VYSMTWVRQA PGKGLEWVSS IGSSGGSTTY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAYRR TGIPRRDAFD IWGQGTMVTV 120SSASTKGPSV FPLAPSSKS 139 M198-B12 LCQDIQMTQSPS TLSASVGDRV TITCRASQSI SSWLAWYQQK PGKAPNLLIY KASTLESGVP  60SRFSGSGSGT EFTLTISSLQ PDDFATYYCQ QYNTYWTFGQ GTKVEIK 107 M198-B12 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS KYSMAWVRQA PGKGLEWVSG IYPSGGRTLY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAYRR TGIPRRDAFD IWGQGTMVTV 120SSASTKGPSV FPLAPSSKS 139 M198-C03 LCQDIQMTQSPS TLSASVGDRV TITCRASQSI SSWLAWYQQK PGKAPNLLIY KASTLESGVP  60SRFSGSGSGT EFTLTISSLQ PDDFATYYCQ QYNTYWTFGQ GTKVEIK 107 M198-C03 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS RYSMSWVRQA PGKGLEWVSG ISPSGGETSY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAYRR TGIPRRDAFD IWGQGTMVTV 120SSASTKGPSV FPLAPSSKS 139 M198-C05 LCQDIQMTQSPS TLSASVGDRV TITCRASQSI SSWLAWYQQK PGKAPNLLIY KASTLESGVP  60SRFSGSGSGT EFTLTISSLQ PDDFATYYCQ QYNTYWTFGQ GTKVEIK 107 M198-C05 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS RYKMYWVRQA PGKGLEWVSV IGPSGGATFY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAYRR TGIPRRDAFD IWGQGTMVTV 120SSASTKGPSV FPLAPSSKS 139 M198-C06 LCQDIQMTQSPS TLSASVGDRV TITCRASQSI SSWLAWYQQK PGKAPNLLIY KASTLESGVP  60SRFSGSGSGT EFTLTISSLQ PDDFATYYCQ QYNTYWTFGQ GTKVEIK 107 M198-C06 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS QYFMHWVRQA PGKGLEWVSY IYPSGGMTEY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAYRR TGIPRRDAFD IWGQGTMVTV 120SSASTKGPSV FPLAPSSKS 139 M198-C09 LCQDIQMTQSPS TLSASVGDRV TITCRASQSI SSWLAWYQQK PGKAPNLLIY KASTLESGVP  60SRFSGSGSGT EFTLTISSLQ PDDFATYYCQ QYNTYWTFGQ GTKVEIK 107 M198-C09 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS IYTMYWVRQA PGKGLEWVSS ISPSGGWTYY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAYRR TGIPRRDAFD IWGQGTMVTV 120SSASTKGPSV FPLAPSSKS 139 M198-C10 LCQDIQMTQSPS TLSASVGDRV TITCRASQSI SSWLAWYQQK PGKAPNLLIY KASTLESGVP  60SRFSGSGSGT EFTLTISSLQ PDDFATYYCQ QYNTYWTFGQ GTKVEIK 107 M198-C10 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS HYMGMNWVRQ APGKGLEWVS SIVPSGGWTQ  60YADSVKGRFT ISRDNSKNTL YLQMNSLRAE DTAVYYCAYR RTGIPRRDAF DIWGQGTMVT 120VSSASTKGPS VFPLAPSSKS 140 M198-D12 LCQDIQMTQSPS TLSASVGDRV TITCRASQSI SSWLAWYQQK PGKAPNLLIY KASTLESGVP  60SRFSGSGSGT EFTLTISSLQ PDDFATYYCQ QYNTYWTFGQ GTKVEIK 107 M198-D12 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS LYVMYWVRQA PGKGLEWVSY IVPSGGPTAY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAYRR TGIPRRDAFD IWGQGTMVTV 120SSASTKGPSV FPLAPSSKS 139 M198-E02 LCQDIQMTQSPS TLSASVGDRV TITCRASQSI SSWLAWYQQK PGKAPNLLIY KASTLESGVP  60SRFSGSGSGT EFTLTISSLQ PDDFATYYCQ QYNTYWTFGQ GTKVEIK 107 M198-E02 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS IYSMYWVRQA PGKGLEWVSY IRPSGGNTKY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAYRR TGIPRRDAFD IWGQGTMVTV 120SSASTKGPSV FPLAPSSKS 139 M198-E09 LCQDIQMTQSPS TLSASVGDRV TITCRASQSI SSWLAWYQQK PGKAPNLLIY KASTLESGVP  60SRFSGSGSGT EFTLTISSLQ PDDFATYYCQ QYNTYWTFGQ GTKVEIK 107 M198-E09 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS IYDMNWVRQA PGKGLEWVSS IYPSGGRTRY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAYRR TGIPRRDAFD IWGQGTMVTV 120SSASTKGPSV FPLAPSSKS 139 M198-E11 LCQDIQMTQSPS TLSASVGDRV TITCRASQSI SSWLAWYQQK PGKAPNLLIY KASTLESGVP  60SRFSGSGSGT EFTLTISSLQ PDDFATYYCQ QYNTYWTFGQ GTKVEIK 107 M198-E11 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS TYSMYWVRQA PGKGLEWVSS IYPSGGLTWY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAYRR TGIPRRDAFD IWGQGTMVTV 120SSASTKGPSV FPLAPSSKS 139 M198-F04 LCQDIQMTQSPS TLSASVGDRV TITCRASQSI SSWLAWYQQK PGKAPNLLIY KASTLESGVP  60SRFSGSGSGT EFTLTISSLQ PDDFATYYCQ QYNTYWTFGQ GTKVEIK 107 M198-F04 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS IYDMWWVRQA PGKGLEWVSS IRPSGGITKY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAYRR TGIPRRDAFD IWGQGTMVTV 120SSASTKGPSV FPLAPSSKS 139 M198-F08 LCQDIQMTQSPS TLSASVGDRV TITCRASQSI SSWLAWYQQK PGKAPNLLIY KASTLESGVP  60SRFSGSGSGT EFTLTISSLQ PDDFATYYCQ QYNTYWTFGQ GTKVEIK 107 M198-F08 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS VYSMWWVRQA PGKGLEWVSS ISSSGGMTEY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAYRR TGIPRRDAFD IWGQGTMVTV  120SSASTKGPSV FPLAPSSKS 139 M198-F09 LCQDIQMTQSPS TLSASVGDRV TITCRASQSI SSWLAWYQQK PGKAPNLLIY KASTLESGVP  60SRFSGSGSGT EFTLTISSLQ PDDFATYYCQ QYNTYWTFGQ GTKVEIK 107 M198-F09 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS VYNMHWVRQA PGKGLEWVSS IYPSGGMTYY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAYRR TGIPRRDAFD IWGQGTMVTV 120SSASTKGPSV FPLAPSSKS 139 M198-F12 LCQDIQMTQSPS TLSASVGDRV TITCRASQSI SSWLAWYQQK PGKAPNLLIY KASTLESGVP  60SRFSGSGSGT EFTLTISSLQ PDDFATYYCQ QYNTYWTFGQ GTKVEIK 107 M198-F12 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS IYTMMWVRQA PGKGLEWVSS IWSSGGQTKY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAYRR TGIPRRDAFD IWGQGTMVTV 120SSASTKGPSV FPLAPSSKS 139 M198-G03 LCQDIQMTQSPS TLSASVGDRV TITCRASQSI SSWLAWYQQK PGKAPNLLIY KASTLESGVP  60SRFSGSGSGT EFTLTISSLQ PDDFATYYCQ QYNTYWTFGQ GTKVEIK 107 M198-G03 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS QYTMVWVRQA PGKGLEWVSW IYSSRANYAD  60SVKGRFTISR DNSKNTLYLQ MNSLRAEDTA VYYCAYRRTG IPRRDAFDIW GQGTMVTVSS 120ASTKGPSVFP LAPSSKS 137 M198-G07 LCQDIQMTQSPS TLSASVGDRV TITCRASQSI SSWLAWYQQK PGKAPNLLIY KASTLESGVP  60SRFSGSGSGT EFTLTISSLQ PDDFATYYCQ QYNTYWTFGQ GTKVEIK 107 M198-G07 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS IYDMTWVRQA PGKGLEWVSS IYPSGGQTIY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAYRR TGIPRRDAFD IWGQGTMVTV 120SSASTKGPSV FPLAPSSKS 139 M198-H02 LCQDIQMTQSPS TLSASVGDRV TITCRASQSI SSWLAWYQQK PGKAPNLLIY KASTLESGVP  60SRFSGSGSGT EFTLTISSLQ PDDFATYYCQ QYNTYWTFGQ GTKVEIK 107 M198-H02 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS PYNMYWVRQA PGKGLEWVSW IVPGGVTKYA  60DSVKGRFTIS RDNSKNTLYL QMNSLRAEDT AVYYCAYRRT GIPRRDAFDI WGQGTMVTVS 120SASTKGPSVF PLAPSSKS 138 M198-H08 LCQDIQMTQSPS TLSASVGDRV TITCRASQSI SSWLAWYQQK PGKAPNLLIY KASTLESGVP  60SRFSGSGSGT EFTLTISSLQ PDDFATYYCQ QYNTYWTFGQ GTKVEIK 107 M198-H08 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS IYDMYWVRQA PGKGLEWVSS IGPSGGPTAY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAYRR TGIPRRDAFD IWGQGTMVTV 120SSASTKGPSV FPLAPSSKS 139 M198-H09 LCQDIQMTQSPS TLSASVGDRV TITCRASQSI SSWLAWYQQK PGKAPNLLIY KASTLESGVP  60SRFSGSGSGT EFTLTISSLQ PDDFATYYCQ QYNTYWTFGQ GTKVEIK 107 M198-H09 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS IYLMIWVRQA PGKGLEWVSY IGPSGGPTEY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAYRR TGIPRRDAFD IWGQGTMVTV 120SSASTKGPSV FPLAPSSKS 139 M199-A08 LCQDIQMTQSPS TLSASVGDRV TITCRASQSI SSWLAWYQQK PGKAPNLLIY KASTLESGVP  60SRFSGSGSGT EFTLTISSLQ PDDFATYYCQ QYNTYWTFGQ GTKVEIK 107 M199-A08 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS HYIMMWVRQA PGKGLEWVSG IYSSGGITVY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAYRR IGVPRRDEFD IWGQGTMVTV 120SSASTKGPSV FPLAPSSKS 139 M199-A11 LCQDIQMTQSPS TLSASVGDRV TITCRASQSI SSWLAWYQQK PGKAPNLLIY KASTLESGVP  60SRFSGSGSGT EFTLTISSLQ PDDFATYYCQ QYNTYWTFGQ GTKVEIK 107 M199-A11 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS HYIMMWVRQA PGKGLEWVSG IYSSGGITVY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAYRR RGIPRRDAFD IWGQGTMVTV 120SSASTKGPSV FPLAPSSKS 139 M199-B01 LCQDIQMTQSPS TLSASVGDRV TITCRASQSI SSWLAWYQQK PGKAPNLLIY KASTLESGVP  60SRFSGSGSGT EFTLTISSLQ PDDFATYYCQ QYNTYWTFGQ GTKVEIK 107 M199-B01 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS HYIMMWVRQA PGKGLEWVSG IYSSGGITVY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAFRR TGIPRRDAFD NWGQGTMVTV 120SSASTKGPSV FPLAPSSKS 139 M200-A10 LCQDIQMTQSPS TLSASVGDRV TITCRASQSI SSWLAWYQQK PGKAPNLLIY KASTLESGVP  60SRFSGSGSGT EFTLTISSLQ PDDFATYYCQ QYNTYWTFGQ GTKVEIK 107 M200-A10 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS HYIMMWVRQA PGKGLEWVSG IYSSGGITVY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAYRR TGVPRRDSFD IWGQGTMVTV 120SSASTKGPSV FPLAPSSKS 139 M200-B01 LCQDIQMTQSPS TLSASVGDRV TITCRASQSI SSWLAWYQQK PGKAPNLLIY KASTLESGVP  60SRFSGSGSGT EFTLTISSLQ PDDFATYYCQ QYNTYWTFGQ GTKVEIK 107 M200-B01 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS HYIMMWVRQA PGKGLEWVSG IYSSGGITVY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAYRR TGIPRRDAFD SWGQGTMVTV 120SSASTKGPSV FPLAPSSKS 139 M200-D03 LCQDIQMTQSPS TLSASVGDRV TITCRASQSI SSWLAWYQQK PGKAPNLLIY KASTLESGVP  60SRFSGSGSGT EFTLTISSLQ PDDFATYYCQ QYNTYWTFGQ GTKVEIK 107 M200-D03 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS HYIMMWVRQA PGKGLEWVSG IYSSGGITVY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAWRR IGVPRRDSFD MWGQGTMVTV 120SSASTKGPSV FPLAPSSKS 139 M200-E11 LCQDIQMTQSPS TLSASVGDRV TITCRASQSI SSWLAWYQQK PGKAPNLLIY KASTLESGVP  60SRFSGSGSGT EFTLTISSLQ PDDFATYYCQ QYNTYWTFGQ GTKVEIK 107 M200-E11 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS HYIMMWVRQA PGKGLEWVSG IYSSGGITVY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAYRR TGVPRRDAFD NWGQGTMVTV 120SSASTKGPSV FPLAPSSKS 139 M200-F01 LCQDIQMTQSPS TLSASVGDRV TITCRASQSI SSWLAWYQQK PGKAPNLLIY KASTLESGVP  60SRFSGSGSGT EFTLTISSLQ PDDFATYYCQ QYNTYWTFGQ GTKVEIK 107 M200-F01 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS HYIMMWVRQA PGKGLEWVSG IYSSGGITVY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAYRR MGIPRRNAFD IWGQGTMVTV 120SSASTKGPSV FPLAPSSKS 139 M200-H07 LCQDIQMTQSPS TLSASVGDRV TITCRASQSI SSWLAWYQQK PGKAPNLLIY KASTLESGVP  60SRFSGSGSGT EFTLTISSLQ PDDFATYYCQ QYNTYWTFGQ GTKVEIK 107 M200-H07 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS HYIMMWVRQA PGKGLEWVSG IYSSGGITVY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAYRR TGVPRRNAFD NWGQGTMVTV 120SSASTKGPSV FPLAPSSKS 139 M201-A06 LCQDIQMTQSPS TLSASVGDRV TITCRASQSI SSWLAWYQQK PGKAPNLLIY KASTLESGVP  60SRFSGSGSGT EFTLTISSLQ PDDFATYYCQ QYNTYWTFGQ GTKVEIK 107 M201-A06 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS HYIMMWVRQA PGKGLEWVSG IYSSGGITVY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAYRR TGIPRRDVFD IWGQGTMVTV 120SSASTKGPSV FPLAPSSKS 139 M201-A07 LCQDIQMTQSPS TLSASVGDRV TITCRASQSI SSWLAWYQQK PGKAPNLLIY KASTLESGVP  60SRFSGSGSGT EFTLTISSLQ PDDFATYYCQ QYNTYWTFGQ GTKVEIK 107 M201-A07 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS HYIMMWVRQA PGKGLEWVSG IYSSGGITVY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAYRR TGVPRRDEFD IWGQGTMVTV 120SSASTKGPSV FPLAPSSKS 139 M201-F11 LCQDIQMTQSPS TLSASVGDRV TITCRASQSI SSWLAWYQQK PGKAPNLLIY KASTLESGVP  60SRFSGSGSGT EFTLTISSLQ PDDFATYYCQ QYNTYWTFGQ GTKVEIK 107 M201-F11 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS HYIMMWVRQA PGKGLEWVSG IYSSGGITVY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAYRR SGIPRRDAFD IWGQGTMVTV 120SSASTKGPSV FPLAPSSKS 139 M201-H06 LCQDIQMTQSPS TLSASVGDRV TITCRASQSI SSWLAWYQQK PGKAPNLLIY KASTLESGVP  60SRFSGSGSGT EFTLTISSLQ PDDFATYYCQ QYNTYWTFGQ GTKVEIK 107 M201-H06 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS HYIMMWVRQA PGKGLEWVSG IYSSGGITVY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAYRR TGVPRRDAFD IWGQGTMVTV 120SSASTKGPSV FPLAPSSKS 139 M201-H08 LCQDIQMTQSPS TLSASVGDRV TITCRASQSI SSWLAWYQQK PGKAPNLLIY KASTLESGVP  60SRFSGSGSGT EFTLTISSLQ PDDFATYYCQ QYNTYWTFGQ GTKVEIK 107 M201-H08 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS HYIMMWVRQA PGKGLEWVSG IYSSGGITVY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAYRR TGVPRRDALD NWGQGTMVTV 120SSASTKGPSV FPLAPSSKS 139 M202-A01 LCQDIQMTQSPS TLSASVGDRV TITCRASQSI SSWLAWYQQK PGKAPNLLIY KASTLESGVP  60SRFSGSGSGT EFTLTISSLQ PDDFATYYCQ QYNTYWTFGQ GTKVEIK 107 M202-A01 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS HYIMMWVRQA PGKGLEWVSG IYSSGGITVY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAYRR IGVPRRDAFD IWGQGTMVTV 120SSASTKGPSV FPLAPSSKS 139 M202-A04 LCQDIQMTQSPS TLSASVGDRV TITCRASQSI SSWLAWYQQK PGKAPNLLIY KASTLESGVP  60SRFSGSGSGT EFTLTISSLQ PDDFATYYCQ QYNTYWTFGQ GTKVEIK 107 M202-A04 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS HYIMMWVRQA PGKGLEWVSG IYSSGGITVY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAYRR KGIPRRDDFD IWGQGTMVTV 120SSASTKGPSV FPLAPSSKS 139 M202-A08 LCQDIQMTQSPS TLSASVGDRV TITCRASQSI SSWLAWYQQK PGKAPNLLIY KASTLESGVP  60SRFSGSGSGT EFTLTISSLQ PDDFATYYCQ QYNTYWTFGQ GTKVEIK 107 M202-A08 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS HYIMMWVRQA PGKGLEWVSG IYSSGGITVY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAYRR TGIPRWDAFD IWGQGTMVTV 120SSASTKGPSV FPLAPSSKS 139 M202-A10 LCQDIQMTQSPS TLSASVGDRV TITCRASQSI SSWLAWYQQK PGKAPNLLIY KASTLESGVP  60SRFSGSGSGT EFTLTISSLQ PDDFATYYCQ QYNTYWTFGQ GTKVEIK 107 M202-A10 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS HYIMMWVRQA PGKGLEWVSG IYSSGGITVY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAFRR TGIPRRDSFD IWGQGTMVTV 120SSASTKGPSV FPLAPSSKS 139 M202-A12 LCQDIQMTQSPS TLSASVGDRV TITCRASQSI SSWLAWYQQK PGKAPNLLIY KASTLESGVP  60SRFSGSGSGT EFTLTISSLQ PDDFATYYCQ QYNTYWTFGQ GTKVEIK 107 M202-A12 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS HYIMMWVRQA PGKGLEWVSG IYSSGGITVY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAYQR TGVPRRDSFN IWGQGTMVTV 120SSASTKGPSV FPLAPSSKS 139 M202-B03 LCQDIQMTQSPS TLSASVGDRV TITCRASQSI SSWLAWYQQK PGKAPNLLIY KASTLESGVP  60SRFSGSGSGT EFTLTISSLQ PDDFATYYCQ QYNTYWTFGQ GTKVEIK 107 M202-B03 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS HYIMMWVRQA PGKGLEWVSG IYSSGGITVY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAYRR TGVPRRDDFD IWGQGTMVTV 120SSASTKGPSV FPLAPSSKS 139 M202-B04 LCQDIQMTQSPS TLSASVGDRV TITCRASQSI SSWLAWYQQK PGKAPNLLIY KASTLESGVP  60SRFSGSGSGT EFTLTISSLQ PDDFATYYCQ QYNTYWTFGQ GTKVEIK 107 M202-B04 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS HYIMMWVRQA PGKGLEWVSG IYSSGGITVY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAYRR SGVPRRDDFD IWGQGTMVTV 120SSASTKGPSV FPLAPSSKS 139 M202-C01 LCQDIQMTQSPS TLSASVGDRV TITCRASQSI SSWLAWYQQK PGKAPNLLIY KASTLESGVP  60SRFSGSGSGT EFTLTISSLQ PDDFATYYCQ QYNTYWTFGQ GTKVEIK 107 M202-C01 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS HYIMMWVRQA PGKGLEWVSG IYSSGGITVY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAYRR TGVPRWDDFD IWGQGTMVTV 120SSASTKGPSV FPLAPSSKS 139 M202-C02 LCQDIQMTQSPS TLSASVGDRV TITCRASQSI SSWLAWYQQK PGKAPNLLIY KASTLESGVP  60SRFSGSGSGT EFTLTISSLQ PDDFATYYCQ QYNTYWTFGQ GTKVEIK 107 M202-C02 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS HYIMMWVRQA PGKGLEWVSG IYSSGGITVY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAYRR PGVPRRDAFD IWGQGTMVTV 120SSASTKGPSV FPLAPSSKS 139 M202-C09 LCQDIQMTQSPS TLSASVGDRV TITCRASQSI SSWLAWYQQK PGKAPNLLIY KASTLESGVP  60SRFSGSGSGT EFTLTISSLQ PDDFATYYCQ QYNTYWTFGQ GTKVEIK 107 M202-C09 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS HYIMMWVRQA PGKGLEWVSG IYSSGGITVY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAYRR IGVPRRDDFD IWGQGTMVTV 120SSASTKGPSV FPLAPSSKS 139 M202-D09 LCQDIQMTQSPS TLSASVGDRV TITCRASQSI SSWLAWYQQK PGKAPNLLIY KASTLESGVP  60SRFSGSGSGT EFTLTISSLQ PDDFATYYCQ QYNTYWTFGQ GTKVEIK 107 M202-D09 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS HYIMMWVRQA PGKGLEWVSG IYSSGGITVY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAYRR IGVPRRDSFD IWGQGTMVTV 120SSASTKGPSV FPLAPSSKS 139 M202-E03 LCQDIQMTQSPS TLSASVGDRV TITCRASQSI SSWLAWYQQK PGKAPNLLIY KASTLESGVP  60SRFSGSGSGT EFTLTISSLQ PDDFATYYCQ QYNTYWTFGQ GTKVEIK 107 M202-E03 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS HYIMMWVRQA PGKGLEWVSG IYSSGGITVY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAYRR TGIPRRDAFE IWGQGTMVTV 120SSASTKGPSV FPLAPSSKS 139 M202-E06 LCQDIQMTQSPS TLSASVGDRV TITCRASQSI SSWLAWYQQK PGKAPNLLIY KASTLESGVP  60SRFSGSGSGT EFTLTISSLQ PDDFATYYCQ QYNTYWTFGQ GTKVEIK 107 M202-E06 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS HYIMMWVRQA PGKGLEWVSG IYSSGGITVY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAYRR RGVPRRDDFD IWGQGTMVTV 120SSASTKGPSV FPLAPSSKS 139 M202-F06 LCQDIQMTQSPS TLSASVGDRV TITCRASQSI SSWLAWYQQK PGKAPNLLIY KASTLESGVP  60SRFSGSGSGT EFTLTISSLQ PDDFATYYCQ QYNTYWTFGQ GTKVEIK 107 M202-F06 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS HYIMMWVRQA PGKGLEWVSG IYSSGGITVY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAYRR TGVPRWDAFD IWGQGTMVTV 120SSASTKGPSV FPLAPSSKS 139 M202-G03 LCQDIQMTQSPS TLSASVGDRV TITCRASQSI SSWLAWYQQK PGKAPNLLIY KASTLESGVP  60SRFSGSGSGT EFTLTISSLQ PDDFATYYCQ QYNTYWTFGQ GTKVEIK 107 M202-G03 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS HYIMMWVRQA PGKGLEWVSG IYSSGGITVY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAFRR TGVPRRDSFE IWGQGTMVTV 120SSASTKGPSV FPLAPSSKS 139 M202-H03 LCQDIQMTQSPS TLSASVGDRV TITCRASQSI SSWLAWYQQK PGKAPNLLIY KASTLESGVP  60SRFSGSGSGT EFTLTISSLQ PDDFATYYCQ QYNTYWTFGQ GTKVEIK 107 M202-H03 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS HYIMMWVRQA PGKGLEWVSG IYSSGGITVY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAFRR TGVPRWDDFD IWGQGTMVTV 120SSASTKGPSV FPLAPSSKS 139 M202-H05 LCQDIQMTQSPS TLSASVGDRV TITCRASQSI SSWLAWYQQK PGKAPNLLIY KASTLESGVP  60SRFSGSGSGT EFTLTISSLQ PDDFATYYCQ QYNTYWTFGQ GTKVEIK 107 M202-H05 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS HYIMMWVRQA PGKGLEWVSG IYSSGGITVY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAFRR TGVPRRDVFD IWGQGTMVTV 120SSASTKGPSV FPLAPSSKS 139

TABLE 9Sequences of Antibodies Obtained from CDR1/2 and CDR3 Spiking Affinity MaturationLibraries Based on X63-G06 (SEQ ID NOs: 1537-1725) HV- CDR1 % human (SEQinhi- pKal LV-CDR2 ID bition Ki, LV-CDR1 (SEQ ID LV-CDR3 NOs: HV-CDR2HV-CDR3 Antibody at app (SEQ ID NO: (SEQ ID 1540- (SEQ ID NOs:(SEQ ID NOs: I.D. 10 nM (nM) NO: 1537) 1538) NO: 1539) 1601 1602-1663)1664-1725) M209-F04 97.6 0.09 RTSQFVNSNYLA GASSRAT QQSSRTPWT HYLMTYISPSGGHTIYADSVKG VARGIAARSRTSYLDq M209-C11 96.2 0.14 RTSQFVNSNYLAGASSRAT QQSSRTPWT HYLMT YISPSGGHTIYADSVKG VGQGIRGRSRTSYFAq M206-H08 96.00.17 RTSQFVNSNYLA GASSRAT QQSSRTPWT DYMMA SIVPSGGHTHYADSVKGVARGIAARSRTSYFDY M210-C12 95.6 0.16 RTSQFVNSNYLA GASSRAT QQSSRTPWT HYLMTYISPSGGHTIYADSVKG VAQGIAARSRTSSVDq M208-F04 95.4 0.2 RTSQFVNSNYLAGASSRAT QQSSRTPWT HYLMT YISPSGGHTIYADSVKG VARGIAARSRTSFFDY M206-B10 94.70.3 RTSQFVNSNYLA GASSRAT QQSSRTPWT qYLMA SIYPSGGWTKYADSVKGVARGIAARSRTSYFDY M208-H02 94.4 0.2 RTSQFVNSNYLA GASSRAT QQSSRTPWT HYLMTYISPSGGHTIYADSVKG VARGIASRSRTRYCDY M210-G04 94.2 0.3 RTSQFVNSNYLAGASSRAT QQSSRTPWT HYLMT YISPSGGHTIYADSVKG VATGIVARSRTRYFDq M210-H06 93.80.2 RTSQFVNSNYLA GASSRAT QQSSRTPWT HYLMT YISPSGGHTIYADSVKGVARGIAARSRTRYFDY M208-E10 93.7 0.09 RTSQFVNSNYLA GASSRAT QQSSRTPWT HYLMTYISPSGGHTIYADSVKG VAQGISARSRTSYFDY M209-B09 93.5 0.2 RTSQFVNSNYLAGASSRAT QQSSRTPWT HYLMT YISPSGGHTIYADSVKG VAQGIVARSRTSYLHq M209-C12 93.4RTSQFVNSNYLA GASSRAT QQSSRTPWT HYLMT YISPSGGHTIYADSVKG VGRGIAARSRTSqLDYM208-G03 93.4 0.3 RTSQFVNSNYLA GASSRAT QQSSRTPWT HYLMT YISPSGGHTIYADSVKGVARGIAARSRTSYLDY M206-A06 93.0 RTSQFVNSNYLA GASSRAT QQSSRTPWT NYMMGSISPSGGLTKYADSVKG VARGIAARSRTSYFDY M210-H07 92.8 0.4 RTSQFVNSNYLAGASSRAT QQSSRTPWT HYLMT YISPSGGHTIYADSVKG VARGIAARSRTRYFDq M206-F01 92.60.2 RTSQFVNSNYLA GASSRAT QQSSRTPWT GYMMV RISPSGGPTIYADSVKGVARGIAARSRTSYFDY M208-F10 92.5 0.2 RTSQFVNSNYLA GASSRAT QQSSRTPWT HYLMTYISPSGGHTIYADSVKG VARGIAARSRTSYFDq M209-E02 92.4 0.3 RTSQFVNSNYLAGASSRAT QQSSRTPWT HYLMT YISPSGGHTIYADSVKG VARGIAARSRTILLDq M208-C06 91.70.4 RTSQFVNSNYLA GASSRAT QQSSRTPWT HYLMT YISPSGGHTIYADSVKGVARGIAARSRTSFIDY M205-D04 91.5 0.4 RTSQFVNSNYLA GASSRAT QQSSRTPWT TYKMqSISPSGGPTNYADSVKG VARGIAARSRTSYFDY M210-G10 91.2 0.4 RTSQFVNSNYLAGASSRAT QQSSRTPWT HYLMT YISPSGGHTIYADSVKG VARGIAARSRTSYLDF M207-A04 90.9RTSQFVNSNYLA GASSRAT QQSSRTPWT HYLMT YISPSGGHTIYADSVKG VARGIAARSRTRSFDYM210-B02 90.9 0.2 RTSQFVNSNYLA GASSRAT QQSSRTPWT HYLMT YISPSGGHTIYADSVKGVARGIAARSRTSYFNq M208-B01 90.1 RTSQFVNSNYLA GASSRAT QQSSRTPWT HYLMTYISPSGGHTIYADSVKG VARGIAARSRTSFFDq M209-G07 89.8 RTSQFVNSNYLA GASSRATQQSSRTPWT HYLMT YISPSGGHTIYADSVKG VARGIAARSRTSYFDT M204-A02 89.5RTSQFVNSNYLA GASSRAT QQSSRTPWT DYMMT YISPSGGLTSYADSVKG VARGIAARSRTSYFDYM206-H01 87.6 RTSQFVNSNYLA GASSRAT QQSSRTPWT EYMMV RISPSGGTTEYADSVKGVARGIAARSRTSYFDY M209-B11 87.3 RTSQFVNSNYLA GASSRAT QQSSRTPWT HYLMTYISPSGGHTIYADSVKG VARGIAARSRTRYIDq M206-F09 86.8 RTSQFVNSNYLA GASSRATQQSSRTPWT VYMMS SIVPSGGSTTYADSVKG VARGIAARSRTSYFDY M209-C02 86.8RTSQFVNSNYLA GASSRAT QQSSRTPWT HYLMT YISPSGGHTIYADSVKG VARGIAYRRRTSYFDYM208-G02 86.7 RTSQFVNSNYLA GASSRAT QQSSRTPWT HYLMT YISPSGGHTIYADSVKGVARGIADRSRTSYSDY M205-C11 86.5 RTSQFVNSNYLA GASSRAT QQSSRTPWT QYMMMRISPSGGSTLYADSVKG VARGIAARSRTSYFDY M205-H08 85.9 RTSQFVNSNYLA GASSRATQQSSRTPWT DYMMM SIVPSGGHTqYADSVKG VARGIAARSRTSYFDY M210-H01 85.5RTSQFVNSNYLA GASSRAT QQSSRTPWT HYLMT YISPSGGHTIYADSVKG VARGIAARSRNSqQDYM209-D12 85.4 RTSQFVNSNYLA GASSRAT QQSSRTPWT HYLMT YISPSGGHTIYADSVKGVARGIAARSRTSYFDq M209-H09 85.3 RTSQFVNSNYLA GASSRAT QQSSRTPWT HYLMTYISPSGGHTIYADSVKG VARGIAARSRTVYFDH M204-E12 84.1 RTSQFVNSNYLA GASSRATQQSSRTPWT TYMMq YIGPSGGKTDYADSVKG VARGIAARSRTSYFDY M209-H03 82.6RTSQFVNSNYLA GASSRAT QQSSRTPWT HYLMT YISPSGGHTIYADSVKG VAQGIAARSRTTqFDYM206-H05 82.5 RTSQFVNSNYLA GASSRAT QQSSRTPWT GYKMq SISPSGGITMYADSVKGVARGIAARSRTSYFDY M209-D03 80.4 RTSQFVNSNYLA GASSRAT QQSSRTPWT HYLMTYISPSGGHTIYADSVKG VGRGIAARSRTSFFDq M205-A02 80.3 RTSQFVNSNYLA GASSRATQQSSRTPWT TYLMA GIVSSGGRTLYADSVKG VARGIAARSRTSYFDY M208-A10 78.5RTSQFVNSNYLA GASSRAT QQSSRTPWT HYLMT YISPSGGHTIYADSVKG VARGIAARSRTSqFDHM205-E11 78.2 RTSQFVNSNYLA GASSRAT QQSSRTPWT NYTMG SISPSGGKTDYADSVKGVARGIAARSRTSYFDY M206-E02 77.6 RTSQFVNSNYLA GASSRAT QQSSRTPWT EYMMMVISPSGGQTHYADSVKG VARGIAARSRTSYFDY M205-H01 77.1 RTSQFVNSNYLA GASSRATQQSSRTPWT NYTMQ YISPSGGYTGYADSVKG VARGIAARSRTSYFDY M207-A02 76.6RTSQFVNSNYLA GASSRAT QQSSRTPWT HYLMT YISPSGGHTIYADSVKG VARGIAARSRTINLDYM209-H07 76.1 RTSQFVNSNYLA GASSRAT QQSSRTPWT HYLMT YISPSGGHTIYADSVKGVARGIAARqRTSYYDY M209-G01 74.8 RTSQFVNSNYLA GASSRAT QQSSRTPWT HYLMTYISPSGGHTIYADSVKG VAgGISGRSRLSYVDY M210-A06 74.8 RTSQFVNSNYLA GASSRATQQSSRTPWT HYLMT YISPSGGHTIYADSVKG VARGIAARSRTSqFDY M209-D02 74.7RTSQFVNSNYLA GASSRAT QQSSRTPWT HYLMT YISPSGGHTIYADSVKG VARGITARSRTSYFDDM205-B04 71.1 RTSQFVNSNYLA GASSRAT QQSSRTPWT NYDMI SISSSGGTTKYADSVKGVARGIAARSRTSYFDY M203-A03 69.1 RTSQFVNSNYLA GASSRAT QQSSRTPWT VYMMISISPSGGQTTYADSVKG VARGIAARSRTSYFDY M209-E03 68.8 RTSQFVNSNYLA GASSRATQQSSRTPWT HYLMT YISPSGGHTIYADSVKG qARGIAARSRTSYFDY M207-A01 67.2RTSQFVNSNYLA GASSRAT QQSSRTPWT HYLMT YISPSGGHTIYADSVKG VARGISARSRTSCFDYM206-C03 65.5 RTSQFVNSNYLA GASSRAT QQSSRTPWT qYMMV SIYSSGGNTPYADSVKGVARGIAARSRTSYFDY M207-C05 61.4 RTSQFVNSNYLA GASSRAT QQSSRTPWT HYLMTYISPSGGHTIYADSVKG VGRGIAARSRTSYFDK M205-A12 58.8 RTSQFVNSNYLA GASSRATQQSSRTPWT QYDMI YISSSGGFTRYADSVKG VARGIAARSRTSYFDY M205-F03 58.6RTSQFVNSNYLA GASSRAT QQSSRTPWT SqQMV YISPSGGNTYYADSVKG VARGIAARSRTSYFDYM203-A01 51.4 RTSQFVNSNYLA GASSRAT QQSSRTPWT NYLMA WIVPSGGYTEYADSVKGVARGIAARSRTSYFDY M209-B01 47.0 RTSQFVNSNYLA GASSRAT QQSSRTPWT HYLMTYISPSGGHTIYADSVKG VARGIVARSRTSNFDq M208-D12 43.7 RTSQFVNSNYLA GASSRATQQSSRTPWT HYLMT YISPSGGHTIYADSVKG LARGIAARSRTSYqDI M206-H04 19.0RTSQFVNSNYLA GASSRAT QQSSRTPWT SYMMV SISPSGGYTIqADSVKG VARGIAARSRTSYFDY

Amino acid sequences of light chain (LC) and heavy chain (HC) variabledomain of pKal antibodies obtained from CDR1/2 and CDR3 spiking affinitymaturation libraries based on X63-G06. (SEQ ID NOs:1726-1849)

M203-A01 LCQDIQMTQSPG TLSLSPGERA TLSCRTSQFV NSNYLAWYQQ TPGQAPRLLI YGASSRATGI  60PDRFSGTGYG TDFTLTISRL EPEDYGTYYC QQSSRTPWTF GQGTRVEIK 109 M203-A01 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS NYLMAWVRQA PGKGLEWVSW IVPSGGYTEY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARVA RGIAARSRTS YFDYWGQGTL 120VTVSSASTKG PSVFPLAPSS KS 142 M203-A03 LCQDIQMTQSPG TLSLSPGERA TLSCRTSQFV NSNYLAWYQQ TPGQAPRLLI YGASSRATGI  60PDRFSGTGYG TDFTLTISRL EPEDYGTYYC QQSSRTPWTF GQGTRVEIK 109 M203-A03 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS VYMMIWVRQA PGKGLEWVSS ISPSGGQTTY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARVA RGIAARSRTS YFDYWGQGTL 120VTVSSASTKG PSVFPLAPSS KS 142 M204-A02 LCQDIQMTQSPG TLSLSPGERA TLSCRTSQFV NSNYLAWYQQ TPGQAPRLLI YGASSRATGI  60PDRFSGTGYG TDFTLTISRL EPEDYGTYYC QQSSRTPWTF GQGTRVEIK 109 M204-A02 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS DYMMTWVRQA PGKGLqWVSY ISPSGGLTSY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARVA RGIAARSRTS YFDYWGQGTL 120VTVSSASTKG PSVFPLAPSS KS 142 M204-E12 LCQDIQMTQSPG TLSLSPGERA TLSCRTSQFV NSNYLAWYQQ TPGQAPRLLI YGASSRATGI  60PDRFSGTGYG TDFTLTISRL EPEDYGTYYC QQSSRTPWTF GQGTRVEIK 109 M204-E12 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS TYMMqWVRQA PGKGLEWVSY IGPSGGKTDY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARVA RGIAARSRTS YFDYWGQGTL 120VTVSSASTKG PSVFPLAPSS KS 142 M205-A02 LCQDIQMTQSPG TLSLSPGERA TLSCRTSQFV NSNYLAWYQQ TPGQAPRLLI YGASSRATGI  60PDRFSGTGYG TDFTLTISRL EPEDYGTYYC QQSSRTPWTF GQGTRVEIK 109 M205-A02 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS TYLMAWVRQA PGKGLEWVSG IVSSGGRTLY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARVA RGIAARSRTS YFDYWGQGTL 120VTVSSASTKG PSVFPLAPSS KS 142 M205-A12 LCQDIQMTQSPG TLSLSPGERA TLSCRTSQFV NSNYLAWYQQ TPGQAPRLLI YGASSRATGI  60PDRFSGTGYG TDFTLTISRL EPEDYGTYYC QQSSRTPWTF GQGTRVEIK 109 M205-A12 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS QYDMIWVRQA PGKGLEWVSY ISSSGGFTRY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARVA RGIAARSRTS YFDYWGQGTL 120VTVSSASTKG PSVFPLAPSS KS 142 M205-B04 LCQDIQMTQSPG TLSLSPGERA TLSCRTSQFV NSNYLAWYQQ TPGQAPRLLI YGASSRATGI  60PDRFSGTGYG TDFTLTISRL EPEDYGTYYC QQSSRTPWTF GQGTRVEIK 109 M205-B04 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS NYDMIWVRQA PGKGLEWVSS ISSSGGTTKY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARVA RGIAARSRTS YFDYWGQGTL 120VTVSSASTKG PSVFPLAPSS KS 142 M205-C11 LCQDIQMTQSPG TLSLSPGERA TLSCRTSQFV NSNYLAWYQQ TPGQAPRLLI YGASSRATGI  60PDRFSGTGYG TDFTLTISRL EPEDYGTYYC QQSSRTPWTF GQGTRVEIK 109 M205-C11 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS QYMMMWVRQA PGKGLEWVSR ISPSGGSTLY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARVA RGIAARSRTS YFDYWGQGTL 120VTVSSASTKG PSVFPLAPSS KS 142 M205-D04 LCQDIQMTQSPG TLSLSPGERA TLSCRTSQFV NSNYLAWYQQ TPGQAPRLLI YGASSRATGI  60PDRFSGTGYG TDFTLTISRL EPEDYGTYYC QQSSRTPWTF GQGTRVEIK 109 M205-D04 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS TYKMqWVRQA PGKGLEWVSS ISPSGGPTNY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARVA RGIAARSRTS YFDYWGQGTL 120VTVSSASTKG PSVFPLAPSS KS 142 M205-E11 LCQDIQMTQSPG TLSLSPGERA TLSCRTSQFV NSNYLAWYQQ TPGQAPRLLI YGASSRATGI  60PDRFSGTGYG TDFTLTISRL EPEDYGTYYC QQSSRTPWTF GQGTRVEIK 109 M205-E11 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS NYTMGWVRQA PGKGLEWVSS ISPSGGKTDY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARVA RGIAARSRTS YFDYWGQGTL 120VTVSSASTKG PSVFPLAPSS KS 142 M205-F03 LCQDIQMTQSPG TLSLSPGERA TLSCRTSQFV NSNYLAWYQQ TPGQAPRLLI YGASSRATGI  60PDRFSGTGYG TDFTLTISRL EPEDYGTYYC QQSSRTPWTF GQGTRVEIK 109 M205-F03 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS SqQMVWVRQA PGKGLEWVSY ISPSGGNTYY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARVA RGIAARSRTS YFDYWGQGTL 120VTVSSASTKG PSVFPLAPSS KS 142 M205-H01 LCQDIQMTQSPG TLSLSPGERA TLSCRTSQFV NSNYLAWYQQ TPGQAPRLLI YGASSRATGI  60PDRFSGTGYG TDFTLTISRL EPEDYGTYYC QQSSRTPWTF GQGTRVEIK 109 M205-H01 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS NYTMQWVRQA PGKGLqWVSY ISPSGGYTGY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARVA RGIAARSRTS YFDYWGQGTL 120VTVSSASTKG PSVFPLAPSS KS 142 M205-H08 LCQDIQMTQSPG TLSLSPGERA TLSCRTSQFV NSNYLAWYQQ TPGQAPRLLI YGASSRATGI  60PDRFSGTGYG TDFTLTISRL EPEDYGTYYC QQSSRTPWTF GQGTRVEIK 109 M205-H08 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS DYMMMWVRQA PGKGLEWVSS IVPSGGHTqY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARVA RGIAARSRTS YFDYWGQGTL 120VTVSSASTKG PSVFPLAPSS KS 142 M206-A06 LCQDIQMTQSPG TLSLSPGERA TLSCRTSQFV NSNYLAWYQQ TPGQAPRLLI YGASSRATGI  60PDRFSGTGYG TDFTLTISRL EPEDYGTYYC QQSSRTPWTF GQGTRVEIK 109 M206-A06 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS NYMMGWVRQA PGKGLqWVSS ISPSGGLTKY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARVA RGIAARSRTS YFDYWGQGTL 120VTVSSASTKG PSVFPLAPSS KS 142 M206-B10 LCQDIQMTQSPG TLSLSPGERA TLSCRTSQFV NSNYLAWYQQ TPGQAPRLLI YGASSRATGI  60PDRFSGTGYG TDFTLTISRL EPEDYGTYYC QQSSRTPWTF GQGTRVEIK 109 M206-B10 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS qYLMAWVRQA PGKGLEWVSS IYPSGGWTKY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARVA RGIAARSRTS YFDYWGQGTL 120VTVSSASTKG PSVFPLAPSS KS 142 M206-C03 LCQDIQMTQSPG TLSLSPGERA TLSCRTSQFV NSNYLAWYQQ TPGQAPRLLI YGASSRATGI  60PDRFSGTGYG TDFTLTISRL EPEDYGTYYC QQSSRTPWTF GQGTRVEIK 109 M206-C03 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS qYMMVWVRQA PGKGLEWVSS IYSSGGNTPY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARVA RGIAARSRTS YFDYWGQGTL 120VTVSSASTKG PSVFPLAPSS KS 142 M206-E02 LCQDIQMTQSPG TLSLSPGERA TLSCRTSQFV NSNYLAWYQQ TPGQAPRLLI YGASSRATGI  60PDRFSGTGYG TDFTLTISRL EPEDYGTYYC QQSSRTPWTF GQGTRVEIK 109 M206-E02 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS EYMMMWVRQA PGKGLEWVSV ISPSGGQTHY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARVA RGIAARSRTS YFDYWGQGTL 120VTVSSASTKG PSVFPLAPSS KS 142 M206-F01 LCQDIQMTQSPG TLSLSPGERA TLSCRTSQFV NSNYLAWYQQ TPGQAPRLLI YGASSRATGI  60PDRFSGTGYG TDFTLTISRL EPEDYGTYYC QQSSRTPWTF GQGTRVEIK 109 M206-F01 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS GYMMVWVRQA PGKGLEWVSR ISPSGGPTIY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARVA RGIAARSRTS YFDYWGQGTL 120VTVSSASTKG PSVFPLAPSS KS 142 M206-F09 LCQDIQMTQSPG TLSLSPGERA TLSCRTSQFV NSNYLAWYQQ TPGQAPRLLI YGASSRATGI  60PDRFSGTGYG TDFTLTISRL EPEDYGTYYC QQSSRTPWTF GQGTRVEIK 109 M206-F09 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS VYMMSWVRQA PGKGLEWVSS IVPSGGSTTY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARVA RGIAARSRTS YFDYWGQGTL 120VTVSSASTKG PSVFPLAPSS KS 142 M206-H01 LCQDIQMTQSPG TLSLSPGERA TLSCRTSQFV NSNYLAWYQQ TPGQAPRLLI YGASSRATGI  60PDRFSGTGYG TDFTLTISRL EPEDYGTYYC QQSSRTPWTF GQGTRVEIK 109 M206-H01 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS EYMMVWVRQA PGKGLEWVSR ISPSGGTTEY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARVA RGIAARSRTS YFDYWGQGTL 120VTVSSASTKG PSVFPLAPSS KS 142 M206-H04 LCQDIQMTQSPG TLSLSPGERA TLSCRTSQFV NSNYLAWYQQ TPGQAPRLLI YGASSRATGI  60PDRFSGTGYG TDFTLTISRL EPEDYGTYYC QQSSRTPWTF GQGTRVEIK 109 M206-H04 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS SYMMVWVRQA PGKGLEWVSS ISPSGGYTIq  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARVA RGIAARSRTS YFDYWGQGTL 120VTVSSASTKG PSVFPLAPSS KS 142 M206-H05 LCQDIQMTQSPG TLSLSPGERA TLSCRTSQFV NSNYLAWYQQ TPGQAPRLLI YGASSRATGI  60PDRFSGTGYG TDFTLTISRL EPEDYGTYYC QQSSRTPWTF GQGTRVEIK 109 M206-H05 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS GYKMqWVRQA PGKGLEWVSS ISPSGGITMY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARVA RGIAARSRTS YFDYWGQGTL 120VTVSSASTKG PSVFPLAPSS KS 142 M206-H08 LCQDIQMTQSPG TLSLSPGERA TLSCRTSQFV NSNYLAWYQQ TPGQAPRLLI YGASSRATGI  60PDRFSGTGYG TDFTLTISRL EPEDYGTYYC QQSSRTPWTF GQGTRVEIK 109 M206-H08 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS DYMMAWVRQA PGKGLEWVSS IVPSGGHTHY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARVA RGIAARSRTS YFDYWGQGTL 120VTVSSASTKG PSVFPLAPSS KS 142 M207-A01 LCQDIQMTQSPG TLSLSPGERA TLSCRTSQFV NSNYLAWYQQ TPGQAPRLLI YGASSRATGI  60PDRFSGTGYG TDFTLTISRL EPEDYGTYYC QQSSRTPWTF GQGTRVEIK 109 M207-A01 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS HYLMTWVRQA PGKGLEWVSY ISPSGGHTIY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARVA RGISARSRTS CFDYWGQGTL 120VTVSSASTKG PSVFPLAPSS KS 142 M207-A02 LCQDIQMTQSPG TLSLSPGERA TLSCRTSQFV NSNYLAWYQQ TPGQAPRLLI YGASSRATGI  60PDRFSGTGYG TDFTLTISRL EPEDYGTYYC QQSSRTPWTF GQGTRVEIK 109 M207-A02 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS HYLMTWVRQA PGKGLEWVSY ISPSGGHTIY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TALYYCARVA RGIAARSRTI NLDYWGQGTL 120VTVSSASTKG PSVFPLAPSS KS 142 M207-A04 LCQDIQMTQSPG TLSLSPGERA TLSCRTSQFV NSNYLAWYQQ TPGQAPRLLI YGASSRATGI  60PDRFSGTGYG TDFTLTISRL EPEDYGTYYC QQSSRTPWTF GQGTRVEIK 109 M207-A04 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS HYLMTWVRQA PGKGLEWVSY ISPSGGHTIY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARVA RGIAARSRTR SFDYWGQGTL 120VTVSSASTKG PSVFPLAPSS KS 142 M207-C05 LCQDIQMTQSPG TLSLSPGERA TLSCRTSQFV NSNYLAWYQQ TPGQAPRLLI YGASSRATGI  60PDRFSGTGYG TDFTLTISRL EPEDYGTYYC QQSSRTPWTF GQGTRVEIK 109 R0121-D02 =M0207-C05 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS HYLMTWVRQA PGKGLEWVSY ISPSGGHTIY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARVG RGIAARSRTS YFDKWGQGTL 120VTVSSASTKG PSVFPLAPSS KS 142 M208-A10 LCQDIQMTQSPG TLSLSPGERA TLSCRTSQFV NSNYLAWYQQ TPGQAPRLLI YGASSRATGI 60PDRFSGTGYG TDFTLTISRL EPEDYGTYYC QQSSRTPWTF GQGTRVEIK 109 M208-A10 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS HYLMTWVRQA PGKGLEWVSY ISPSGGHTIY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARVA RGIAARSRTS qFDHWGQGTL 120VTVSSASTKG PSVFPLAPSS KS 142 M208-B01 LCQDIQMTQSPG TLSLSPGERA TLSCRTSQFV NSNYLAWYQQ TPGQAPRLLI YGASSRATGI  60PDRFSGTGYG TDFTLTISRL EPEDYGTYYC QQSSRTPWTF GQGTRVEIK 109 M208-B01 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS HYLMTWVRQA PGKGLEWVSY ISPSGGHTIY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARVA RGIAARSRTS FFDqWGQGTL 120VTVSSASTKG PSVFPLAPSS KS 142 M208-C06 LCQDIQMTQSPG TLSLSPGERA TLSCRTSQFV NSNYLAWYQQ TPGQAPRLLI YGASSRATGI  60PDRFSGTGYG TDFTLTISRL EPEDYGTYYC QQSSRTPWTF GQGTRVEIK 109 M208-C06 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS HYLMTWVRQA PGKGLEWVSY ISPSGGHTIY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARVA RGIAARSRTS FIDYWGQGTL 120VTVSSASTKG PSVFPLAPSS KS 142 M208-D12 LCQDIQMTQSPG TLSLSPGERA TLSCRTSQFV NSNYLAWYQQ TPGQAPRLLI YGASSRATGI  60PDRFSGTGYG TDFTLTISRL EPEDYGTYYC QQSSRTPWTF GQGTRVEIK 109 M208-D12 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS HYLMTWVRQA PGKGLEWVSY ISPSGGHTIY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARLA RGIAARSRTS YqDIWGQGTL 120VTVSSASTKG PSVFPLAPSS KS 142 M208-E10 LCQDIQMTQSPG TLSLSPGERA TLSCRTSQFV NSNYLAWYQQ TPGQAPRLLI YGASSRATGI  60PDRFSGTGYG TDFTLTISRL EPEDYGTYYC QQSSRTPWTF GQGTRVEIK 109 M208-E10 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS HYLMTWVRQA PGKGLEWVSY ISPSGGHTIY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARVA QGISARSRTS YFDYWGQGTL 120VTVSSASTKG PSVFPLAPSS KS 142 M208-F04 LCQDIQMTQSPG TLSLSPGERA TLSCRTSQFV NSNYLAWYQQ TPGQAPRLLI YGASSRATGI  60PDRFSGTGYG TDFTLTISRL EPEDYGTYYC QQSSRTPWTF GQGTRVEIK 109 M208-F04 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS HYLMTWVRQA PGKGLEWVSY ISPSGGHTIY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARVA RGIAARSRTS FFDYWGQGTL 120VTVSSASTKG PSVFPLAPSS KS 142 M208-F10 LCQDIQMTQSPG TLSLSPGERA TLSCRTSQFV NSNYLAWYQQ TPGQAPRLLI YGASSRATGI  60PDRFSGTGYG TDFTLTISRL EPEDYGTYYC QQSSRTPWTF GQGTRVEIK 109 M208-F10 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS HYLMTWVRQA PGKGLEWVSY ISPSGGHTIY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARVA RGIAARSRTS YFDqWGQGTL 120VTVSSASTKG PSVFPLAPSS KS 142 M208-G02 LCQDIQMTQSPG TLSLSPGERA TLSCRTSQFV NSNYLAWYQQ TPGQAPRLLI YGASSRATGI  60PDRFSGTGYG TDFTLTISRL EPEDYGTYYC QQSSRTPWTF GQGTRVEIK 109 M208-G02 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS HYLMTWVRQA PGKGLEWVSY ISPSGGHTIY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARVA RGIADRSRTS YSDYWGQGTL 120VTVSSASTKG PSVFPLAPSS KS 142 M208-G03 LCQDIQMTQSPG TLSLSPGERA TLSCRTSQFV NSNYLAWYQQ TPGQAPRLLI YGASSRATGI  60PDRFSGTGYG TDFTLTISRL EPEDYGTYYC QQSSRTPWTF GQGTRVEIK 109 M208-G03 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS HYLMTWVRQA PGKGLEWVSY ISPSGGHTIY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARVA RGIAARSRTS YLDYWGQGTL 120VTVSSASTKG PSVFPLAPSS KS 142 M208-H02 LCQDIQMTQSPG TLSLSPGERA TLSCRTSQFV NSNYLAWYQQ TPGQAPRLLI YGASSRATGI  60PDRFSGTGYG TDFTLTISRL EPEDYGTYYC QQSSRTPWTF GQGTRVEIK 109 M208-H02 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS HYLMTWVRQA PGKGLEWVSY ISPSGGHTIY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARVA RGIASRSRTR YCDYWGQGTL 120VTVSSASTKG PSVFPLAPSS KS 142 M209-B01 LCQDIQMTQSPG TLSLSPGERA TLSCRTSQFV NSNYLAWYQQ TPGQAPRLLI YGASSRATGI  60PDRFSGTGYG TDFTLTISRL EPEDYGTYYC QQSSRTPWTF GQGTRVEIK 109 M209-B01 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS HYLMTWVRQA PGKGLEWVSY ISPSGGHTIY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARVA RGIVARSRTS NFDqWGQGTL 120VTVSSASTKG PSVFPLAPSS KS 142 M209-B09 LCQDIQMTQSPG TLSLSPGERA TLSCRTSQFV NSNYLAWYQQ TPGQAPRLLI YGASSRATGI  60PDRFSGTGYG TDFTLTISRL EPEDYGTYYC QQSSRTPWTF GQGTRVEIK 109 M209-B09 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS HYLMTWVRQA PGKGLEWVSY ISPSGGHTIY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARVA QGIVARSRTS YLHqWGQGTL 120VTVSSASTKG PSVFPLAPSS KS 142 M209-B11 LCQDIQMTQSPG TLSLSPGERA TLSCRTSQFV NSNYLAWYQQ TPGQAPRLLI YGASSRATGI  60PDRFSGTGYG TDFTLTISRL EPEDYGTYYC QQSSRTPWTF GQGTRVEIK 109 M209-B11 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS HYLMTWVRQA PGKGLEWVSY ISPSGGHTIY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARVA RGIAARSRTR YIDqWGQGTL 120VTVSSASTKG PSVFPLAPSS KS 142 M209-C02 LCQDIQMTQSPG TLSLSPGERA TLSCRTSQFV NSNYLAWYQQ TPGQAPRLLI YGASSRATGI  60PDRFSGTGYG TDFTLTISRL EPEDYGTYYC QQSSRTPWTF GQGTRVEIK 109 M209-C02 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS HYLMTWVRQA PGKGLEWVSY ISPSGGHTIY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARVA RGIAYRRRTS YFDYWGQGTL 120VTVSSASTKG PSVFPLAPSS KS 142 M209-C11 LCQDIQMTQSPG TLSLSPGERA TLSCRTSQFV NSNYLAWYQQ TPGQAPRLLI YGASSRATGI  60PDRFSGTGYG TDFTLTISRL EPEDYGTYYC QQSSRTPWTF GQGTRVEIK 109 M209-C11 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS HYLMTWVRQA PGKGLEWVSY ISPSGGHTIY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAMVG QGIRGRSRTS YFAqWGQGTL 120VTVSSASTKG PSVFPLAPSS KS 142 M209-C12 LCQDIQMTQSPG TLSLSPGERA TLSCRTSQFV NSNYLAWYQQ TPGQAPRLLI YGASSRATGI  60PDRFSGTGYG TDFTLTISRL EPEDYGTYYC QQSSRTPWTF GQGTRVEIK 109 M209-C12 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS HYLMTWVRQA PGKGLEWVSY ISPSGGHTIY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARVG RGIAARSRTS qLDYWGQGTL 120VTVSSASTKG PSVFPLAPSS KS 142 M0209-D02 LCQDIQMTQSPG TLSLSPGERA TLSCRTSQFV NSNYLAWYQQ TPGQAPRLLI YGASSRATGI  60PDRFSGTGYG TDFTLTISRL EPEDYGTYYC QQSSRTPWTF GQGTRVEIK 109 M209-D02 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS HYLMTWVRQA PGKGLEWVSY ISPSGGHTIY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARVA RGITARSRTS YFDDWGQGTL 120VTVSSASTKG PSVFPLAPSS KS 142 M209-D03 LCQDIQMTQSPG TLSLSPGERA TLSCRTSQFV NSNYLAWYQQ TPGQAPRLLI YGASSRATGI  60PDRFSGTGYG TDFTLTISRL EPEDYGTYYC QQSSRTPWTF GQGTRVEIK 109 M209-D03 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS HYLMTWVRQA PGKGLEWVSY ISPSGGHTIY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARVG RGIAARSRTS FFDqWGQGTL 120VTVSSASTKG PSVFPLAPSS KS 142 M209-D12 LCQDIQMTQSPG TLSLSPGERA TLSCRTSQFV NSNYLAWYQQ TPGQAPRLLI YGASSRATGI  60PDRFSGTGYG TDFTLTISRL EPEDYGTYYC QQSSRTPWTF GQGTRVEIK 109 M209-D12 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS HYLMTWVRQA PGKGLEWVSY ISPSGGHTIY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCATVA RGIAARSRTS YFDqWGQGTL 120VTVSSASTKG PSVFPLAPSS KS 142 M209-E02 LCQDIQMTQSPG TLSLSPGERA TLSCRTSQFV NSNYLAWYQQ TPGQAPRLLI YGASSRATGI  60PDRFSGTGYG TDFTLTISRL EPEDYGTYYC QQSSRTPWTF GQGTRVEIK 109 M209-E02 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS HYLMTWVRQA PGKGLEWVSY ISPSGGHTIY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARVA RGIAARSRTI LLDqWGQGTL 120VTVSSASTKG PSVFPLAPSS KS 142 M209-E03 LCQDIQMTQSPG TLSLSPGERA TLSCRTSQFV NSNYLAWYQQ TPGQAPRLLI YGASSRATGI  60PDRFSGTGYG TDFTLTISRL EPEDYGTYYC QQSSRTPWTF GQGTRVEIK 109 M209-E03 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS HYLMTWVRQA PGKGLEWVSY ISPSGGHTIY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARqA RGIAARSRTS YFDYWGQGTL 120VTVSSASTKG PSVFPLAPSS KS 142 M209-F04 LCQDIQMTQSPG TLSLSPGERA TLSCRTSQFV NSNYLAWYQQ TPGQAPRLLI YGASSRATGI  60PDRFSGTGYG TDFTLTISRL EPEDYGTYYC QQSSRTPWTF GQGTRVEIK 109 M209-F04 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS HYLMTWVRQA PGKGLEWVSY ISPSGGHTIY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARVA RGIAARSRTS YLDqWSQGTL 120VTVSSASTKG PSVFPLAPSS KS 142 M209-G01 LCQDIQMTQSPG TLSLSPGERA TLSCRTSQFV NSNYLAWYQQ TPGQAPRLLI YGASSRATGI  60PDRFSGTGYG TDFTLTISRL EPEDYGTYYC QQSSRTPWTF GQGTRVEIK 109 M209-G01 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS HYLMTWVRQA PGKGLEWVSY ISPSGGHTIY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARVA qGISGRSRLS YVDYWGQGTL 120VTVSSASTKG PSVFPLAPSS KS 142 M209-G07 LCQDIQMTQSPG TLSLSPGERA TLSCRTSQFV NSNYLAWYQQ TPGQAPRLLI YGASSRATGI  60PDRFSGTGYG TDFTLTISRL EPEDYGTYYC QQSSRTPWTF GQGTRVEIK 109 M209-G07 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS HYLMTWVRQA PGKGLEWVSY ISPSGGHTIY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARVA RGIAARSRTS YFDTWGQGTL 120VTVSSASTKG PSVFPLAPSS KS 142 M209-H03 LCQDIQMTQSPG TLSLSPGERA TLSCRTSQFV NSNYLAWYQQ TPGQAPRLLI YGASSRATGI  60PDRFSGTGYG TDFTLTISRL EPEDYGTYYC QQSSRTPWTF GQGTRVEIK 109 M209-H03 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS HYLMTWVRQA PGKGLEWVSY ISPSGGHTIY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARVA QGIAARSRTT qFDYWGQGTL 120VTVSSASTKG PSVFPLAPSS KS 142 M209-H07 LCQDIQMTQSPG TLSLSPGERA TLSCRTSQFV NSNYLAWYQQ TPGQAPRLLI YGASSRATGI  60PDRFSGTGYG TDFTLTISRL EPEDYGTYYC QQSSRTPWTF GQGTRVEIK 109 M209-H07 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS HYLMTWVRQA PGKGLEWVSY ISPSGGHTIY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARVA RGIAARqRTS YYDYWGQGTL 120VTVSSASTKG PSVFPLAPSS KS 142 M209-H09 LCQDIQMTQSPG TLSLSPGERA TLSCRTSQFV NSNYLAWYQQ TPGQAPRLLI YGASSRATGI  60PDRFSGTGYG TDFTLTISRL EPEDYGTYYC QQSSRTPWTF GQGTRVEIK 109 M209-H09 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS HYLMTWVRQA PGKGLEWVSY ISPSGGHTIY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARVA RGIAARSRTV YFDHWGQGTL 120VTVSSASTKG PSVFPLAPSS KS 142 M210-A06 LCQDIQMTQSPG TLSLSPGERA TLSCRTSQFV NSNYLAWYQQ TPGQAPRLLI YGASSRATGI  60PDRFSGTGYG TDFTLTISRL EPEDYGTYYC QQSSRTPWTF GQGTRVEIK 109 M210-A06 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS HYLMTWVRQA PGKGLEWVSY ISPSGGHTIY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARVA RGIAARSRTS qFDYWGQGTL 120VTVSSASTKG PSVFPLAPSS KS 142 M210-B02 LCQDIQMTQSPG TLSLSPGERA TLSCRTSQFV NSNYLAWYQQ TPGQAPRLLI YGASSRATGI  60PDRFSGTGYG TDFTLTISRL EPEDYGTYYC QQSSRTPWTF GQGTRVEIK 109 M210-B02 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS HYLMTWVRQA PGKGLEWVSY ISPSGGHTIY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCASVA RGIAARSRTS YFNqWGQGTL 120VTVSSASTKG PSVFPLAPSS KS 142 M210-C12 LCQDIQMTQSPG TLSLSPGERA TLSCRTSQFV NSNYLAWYQQ TPGQAPRLLI YGASSRATGI  60PDRFSGTGYG TDFTLTISRL EPEDYGTYYC QQSSRTPWTF GQGTRVEIK 109 M210-C12 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS HYLMTWVRQA PGKGLEWVSY ISPSGGHTIY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARVA QGIAARSRTS SVDqWGQGTL 120VTVSSASTKG PSVFPLAPSS KS 142 M210-G04 LCQDIQMTQSPG TLSLSPGERA TLSCRTSQFV NSNYLAWYQQ TPGQAPRLLI YGASSRATGI  60PDRFSGTGYG TDFTLTISRL EPEDYGTYYC QQSSRTPWTF GQGTRVEIK 109 M210-G04 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS HYLMTWVRQA PGKGLEWVSY ISPSGGHTIY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARVA TGIVARSRTR YFDqWGQGTL 120VTVSSASTKG PSVFPLAPSS KS 142 M210-G10 LCQDIQMTQSPG TLSLSPGERA TLSCRTSQFV NSNYLAWYQQ TPGQAPRLLI YGASSRATGI  60PDRFSGTGYG TDFTLTISRL EPEDYGTYYC QQSSRTPWTF GQGTRVEIK 109 M210-G10 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS HYLMTWVRQA PGKGLEWVSY ISPSGGHTIY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARVA RGIAARSRTS YLDFWGQGTL 120VTVSSASTKG PSVFPLAPSS KS 142 M210-H01 LCQDIQMTQSPG TLSLSPGERA TLSCRTSQFV NSNYLAWYQQ TPGQAPRLLI YGASSRATGI  60PDRFSGTGYG TDFTLTISRL EPEDYGTYYC QQSSRTPWTF GQGTRVEIK 109 M210-H01 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS HYLMTWVRQA PGKGLEWVSY ISPSGGHTIY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARVA RGIAARSRNS qQDYWGQGTL 120VTVSSASTKG PSVFPLAPSS KS 142 M210-H06 LCQDIQMTQSPG TLSLSPGERA TLSCRTSQFV NSNYLAWYQQ TPGQAPRLLI YGASSRATGI  60PDRFSGTGYG TDFTLTISRL EPEDYGTYYC QQSSRTPWTF GQGTRVEIK 109 M210-H06 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS HYLMTWVRQA PGKGLEWVSY ISPSGGHTIY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARVA RGIAARSRTR YFDYWGQGTL 120VTVSSASTKG PSVFPLAPSS KS 142 M210-H07 LCQDIQMTQSPG TLSLSPGERA TLSCRTSQFV NSNYLAWYQQ TPGQAPRLLI YGASSRATGI  60PDRFSGTGYG TDFTLTISRL EPEDYGTYYC QQSSRTPWTF GQGTRVEIK 109 M210-H07 HCEVQLLESGGG LVQPGGSLRL SCAASGFTFS HYLMTWVRQA PGKGLEWVSY ISPSGGHTIY  60ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARVA RGIAARSRTR YFDqWGQGTL 120VTVSSASTKG PSVFPLAPSS KS 142

Example 6: In Vivo Testing of M162-A04 (IgG) and X101-A01

Bradykinin and other bioactive kinins have been previously implicated incarrageenan-induced edema and inflammatory pain (Sharma J. N. et al.(1998) Inflammopharmacology 6, 9-17; Asano M. et al. (1997) Br JPharmacol 122, 1436-1440; De Campos R. O. et al. (1996) Eur J Pharmacol316, 277-286). Plasma kallikrein and tissue kallikrein 1 are the twoprimary kininogenases in mammals (Schmaier A. H. (2008) IntImmunopharmacol 8, 161-165). M162-A04 (M162-A4) (IgG), a specific plasmakallikrein inhibitor, was tested to determine whether it would beeffective in carrageenan induced edema. The study design is outlined inTable 10. The route of administration (ROA) for the vehicle (PBS), theantibody, and the positive control (indomethacin) was intra-peritoneal(IP) and was given 30 minutes prior to carrageenan injection (0.1 mL ofa 2% carrageenan solution). It is evident from FIG. 2 that antibodydoses at 10 mg/kg and above were equally effective in reducingcarrageenan-induced edema as the positive control (indomethacin).However, the antibody was not effective in reducing carrageenan-inducedthermal hyperalgesia (FIG. 3). The reason for the dissociation betweeneffectiveness in edema and hyperalgesia are not obvious but may be dueto differences in the bioactivity of different kinin metabolites.Lys-desArg9-bradykinin is the most potent agonist of the B1 receptor,which is believed to be primarily involved in pain hypersensitivity(Leeb-Lundberg L. M et al. (2005) Pharmacol Rev 57, 27-77). This kininmetabolite is generated by tissue kallikrein 1, not plasma kallikrein(Schmaier A. H. (2008) Int Immunopharmacol 8, 161-165). This differencein kinin generation and resulting bradykinin receptor activation mayaccount for the unexpected decoupling of edema and hyperalgesia in thismodel.

Another pKal antibody inhibitor X101-A01 was also tested in the CPEmodel using the study design shown in Table 10B. The data obtained inFIG. 14 shows that X101-A01 inhibited edema in a dose-dependent mannerto an extent that is comparable to that of the positive control(indomethacin).

TABLE 10A Carrageenan-Induced Paw Edema Study Design to test M162-A04Timing Num- relative Dose Group ber of Dose to carra- Volume # RatsTreatment (mg/kg) ROA geenan (mL/kg) 1 6 Vehicle N/A IP T −30 20 minutes2 6 559A-M162-A4 3 IP T −30 20 minutes 3 6 559A-M162-A4 10 IP T −30 20minutes 4 6 559A-M162-A4 30 IP T −30 20 minutes 5 6 Indomethacin 5 IP T−30 20 minutes

TABLE 10B Carrageenan-Induced Paw Edema Study Design to Test X101- DoseVol Group Treatment n (mg/Kg) ROA Timing * (mL/Kg) 1 Vehicle 10 N/A IP−30 min 20 2 X101-A01 10 1 IP −30 min 20 3 X101-A01 10 3 IP −30 min 20 4X101-A01 10 10 IP −30 min 20 5 X101-A01 10 30 IP −30 min 20 6Indomethacin 10 5 IP −30 min 20

Example 7: Evaluation of Selected Antibody Inhibitors of PlasmaKallikrein

Evaluation of selected optimized antibodies (X81-B01 and X67-D03) isshown in Table 11. Neither antibody has any putative deamidation,isomerization, or oxidation sites.

TABLE 11 Criteria X81-B01 (IgG) X67-D03 (IgG) <nM Ki, app against humanpKal 0.2 nM 0.1 nM <nM Ki, app against rodent pKal mouse - 11 pM mouse -0.7 nM rat - 0.14 nM rat - 0.34 nM prekallikrein binding no no Specificinhibitor with respect to yes yes fXIa, plasmin, and trypsin Inhibitsbradykinin generation yes yes Inhibits pKal in presence of yes yesprekallikrein Competition for binding with yes yes aprotinin Stabilityin human serum yes nd* *not done; a parental form of this antibody wasshown to be stable in serum

Example 8: Epitope Mapping

The region of pKal bound by selected anti-pKal antibodies wasinvestigated using several methods. First, competition assays were usedto determine whether the antibodies competed for binding to pKal withknown active site-directed inhibitors. Second, antibodies were groupedaccording to whether they were inhibitors or just binders to pKal.Third, epitopes were investigated using synthetic peptides and peptidicstructures based on the sequence and 3-dimensional structure of pKal.These peptidic structures are called “CLIPS” (Chemically Linked Peptideson Scaffolds) and the testing was performed by a fee for service companycalled Pepscan.

Fourth, antibodies were tested for their ability to inhibit pKal fromother species, besides human, where the amino acid sequence of pKal hasbeen determined in order to identify amino acids that may account forthe differences in inhibition.

Competition Assays

Using a BIACORE® SPR assay antibodies of interest were tested forcompetition with a known active site inhibitor of pKal. EPI-KAL2 ispotent (K_(i,app)=0.1 nM) active site inhibitor of pKal and a Kunitzdomain inhibitor based on the first domain of tissue factor pathwayinhibitor (Markland (1996) Iterative optimization of high-affinityprotease inhibitors using phage display. 2. Plasma kallikrein andthrombin. Biochemistry. 35(24):8058-67). Kunitz domains are known activesite inhibitors of serine proteases, such as pKal.

The sequence of EPI-KAL2 is:

(SEQ ID NO: 1850) EAMHSFCAFKADDGPCRAAHPRW FFNIFTRQCEEFSYGGCGGNQNRFESLEECKKMCTRD

(amino acids in italics are those that differ from TFPI)

As shown in FIGS. 8A-8B, the antibodies X81-B01 and X67-D03 werecompeted for binding to pKal in the presence or EPI-KAL2. This resultindicates that these antibodies either bind in vicinity of the activesite or allosteric changes in the conformation of the pKal-EPI-KAL2complex prevent antibody binding.

Antibody Binders vs Inhibitors

As shown in Tables 1 and 2, all the unique antibodies discovered byphage display were characterized as being either pKal inhibitors orbinders but not inhibitors. Antibodies that inhibit the activity of pKaleither bind near the active site and preclude substrate interactions(competitive inhibitors) or that bind away from the active site andinduce allosteric changes in the structure of the active site(noncompetitive inhibitors). Antibodies that bind but do not inhibitpKal are unlikely to bind near the active site and may bind thenon-catalytic domain (i.e. the apple domain). Table 12 categorizesselected antibodies as being either inhibitors or binders of pKal. Alsoshown in Table 12 for the listed antibodies, is a demonstration ofwhether they cross-react with mouse pKal as inhibitors and whether theybind prekallikrein.

TABLE 12 Binding Properties of Selected Anti-pKal Antibodies human mouseCLIPS Ki, app Ki, app Peptide(s) Number Antibody Binding Category (nM)(nM) Identified 1 M6-A06 1) Binder only no no C4 2 M6-D09 2) inhibitor,prekallikrein 5.9 3.9 C1, C5 binder, inhibits mouse and human pKal 3M8-C04 1) Binder only no no 4 M8-G09 1) Binder only no no C1, C4, C6, C75 M29-D09 3) inhibitor, does not bind 0.7 no C1, C4, prekallikrein, doesnot C7 inhibit mouse pKal 6 M35-G04 2) inhibitor, prekallikrein 2.9 8C1, C4 binder, inhibits mouse and human pKal 7 M145- 3) inhibitor, doesnot bind 0.79 800 C1, C4 D11 prekallikrein, weak inhibitor of mouse pKal8 M160- 4) inhibitor of both mouse 5 0.2 C2 G12 and human pKal, does notbind prekallikrein 9 X55-F01 4) inhibitor of both 0.4 2 C2, C3 mouse andhuman pKal, does not bind prekallikrein 10 X73-H09 4) inhibitor, doesnot bind 20 70 C6 prekallikrein, weak inhibitor of human and mouse pKal11 X81-B01 4) inhibitor of both mouse 0.1 0.011 C2, C3, and human pKal,does not C5, C6 bind prekallikrein 12 A2 5) Negative control, does Nobinding No binding No not bind pKal, binds binding streptavidin C1-C7:peptides in pKal identified by CLIPS epitope mapping (see FIGS. 9 and10A-10C). C1 corresponds to positions 55-67 of the catalytic domain, C2to positions 81-94, C3 to positions 101-108, C4 to positions 137-151, C5to positions 162-178, C6 to positions 186-197, and C7 to positions214-217.

Epitope Mapping Using CLIPS

The 11 anti-pKal antibodies listed in Table 12, plus one negativecontrol (A2) were tested for binding to 5000 different synthetic CLIPS(Chemically Linked Peptides on Scaffolds) by Pepscan as described belowin the CLIP METHODS sections. This analysis led to the identification ofpeptide regions in pKal that are likely to be a part of the antibodyepitope for each of the tested antibodies (FIG. 9).

Clips Methods

The linear and CLIPS peptides were synthesized based on the amino acidsequence of the target protein using standard Fmoc-chemistry anddeprotected using trifluoric acid with scavengers. The constrainedpeptides were synthesized on chemical scaffolds in order to reconstructconformational epitopes, using Chemically Linked Peptides on Scaffolds(CLIPS) technology (Timmerman et al. (2007). For example, the singlelooped peptides were synthesized containing a dicysteine, which wascyclized by treating with alpha, alpha′-dibromoxylene and the size ofthe loop was varied by introducing cysteine residues at variablespacing. If other cysteines besides the newly introduced cysteines werepresent, they were replaced by alanine. The side-chains of the multiplecysteines in the peptides were coupled to CLIPS templates by reactingonto credit-card format polypropylene PEPSCAN cards (455 peptideformats/card) with a 0.5 mM solution of CLIPS template such as 1,3-bis(bromomethyl) benzene in ammonium bicarbonate (20 mM, pH7.9)/acetonitrile (1:1(v/v)). The cards were gently shaken in thesolution for 30 to 60 minutes while completely covered in solution.Finally, the cards were washed extensively with excess of H₂O andsonicated in distrupt-buffer containing 1 percent SDS/0.1 percentbeta-mercaptoethanol in PBS (pH 7.2) at 70° C. for 30 minutes, followedby sonication in H₂O for another 45 minutes. The binding of antibody toeach peptide were tested in a PEPSCAN-based ELISA. The 455-well creditcard format polypropylene cards containing the covalently linkedpeptides were incubated with primary antibody solution for exampleconsisting of 1 micrograms/mL diluted in blocking solution called SQ (4%horse serum, 5% ovalbumin (w/v) in PBS/1% Tween or diluted in PBS e.g.,20% SQ) overnight. After washing, the peptides were incubated with a1/1000 dilution of rabbit anti-human antibody peroxidase orgoat-anti-human FAB peroxidase for one hour at 25° C. After washing, theperoxidase substrate 2,2′-azino-di-3-ethylbenzthiazoline sulfonate(ABTS) and 2 microlitres of 3 percent H₂O₂ were added. After one hour,the color development was measured. The color development was quantifiedwith a charge coupled device (CCD)—camera and an image processing system(as firstly described in Slootstra et al., 1996).

Data Calculation

Raw Data: Optical density (Arbitrary OD units) The raw data are opticalvalues obtained by a CCD-camera. The values mostly range from 0 to 3000,a log scale similar to 1 to 3 of a standard 96-well plate elisa-reader.First the CCD-camera makes a picture of the card before peroxidasecoloring and then again a picture after the peroxidase coloring. Thesetwo pictures are subtracted from each other which results in the datawhich is called raw-data. This is copied into the Peplab™ database. Thenthe values are copied to excel and this file is labeled as raw-datafile. One follow-up manipulation is allowed. Sometimes a well containsan air-bubble resulting in a false-positive value, the cards aremanually inspected and any values caused by an air-bubble are scored as0.

Normally assays are not done in replicate (only upon request clientrequest). Replicate tests are usually very similar. In addition, thedataset of thousands of peptides contains many peptides that aresimilar, thus results are never based on recognition of one peptide buton families of similar peptides. If one or a few peptides do not bind,or exhibit lower binding, in a replicate experiment, a different epitopemapping is not normally attributed.

Timmerman et al. (2007). Functional reconstruction and synthetic mimicryof a conformational epitope using CLIPS™ technology. J. Mol. Recognit.20:283-99

Slootstra et al. (1996). Structural aspects of antibody-antigeninteraction revealed through small random peptide libraries, MolecularDiversity, 1, 87-96.

Example 9: Analysis of pKal Sequences from Different Species

All available sequence of pKal were obtained from public databases andaligned using ClustalW and regions were highlighted based on solventaccessibility, contact with an active site Kunitz inhibitor, and thosepeptides identified by CLIPS analysis (FIGS. 10A-10C). Citrated plasmafrom each of these species was obtained and activated using acommercially available prekallikrein activator (from Enzyme ResearchLaboratories) according to the instructions of the manufacturer.Kallikrein activity was then measured in each of the samples in thepresence or absence of X81-B01.

It was found that X81-B01 inhibited pKal from all the species except forpig pKal. Since the CLIPS analysis identified four peptides of pKal thatX81-B01 binds to—C2 (positions 81-94), C3 (positions 101-108), C5(positions 162-178) and C6 (positions 186-197)—differences in the pigpKal sequence that correspond to these peptides were examined toidentify potential amino acids changes that account for the lack ofinhibition of pig pKal by X81-B01. Peptides C2 and C3 are close in thesequence and are both highly similar in sequence among the differentspecies. However, there is a difference at position 479. All the speciesexcept pig, frog, and dog have a serine at position 479. The frog anddog pKal sequence has an alanine and a threonine at position 479,respectively; both of which are considered conservative substitutionsfor a serine. In contrast, the pig pKal sequence has a leucine atposition 479, which is a considerably less conservative substitution fora serine. Peptide C5 in pig pKal is highly similar to the sequences fromthe other species. However, at position 563, only in the pig pKal is ahistidine present (bold in FIG. 10C). This position in all the otherspecies, except frog, is a tyrosine. In the frog pKal, which isinhibited by X81-B01, this position is a threonine. Peptide C6 in pigpKal is again highly similar to the other sequences. However, only inthe pig pKal sequence is position 585 a glutamate (in bold in FIG. 10C).In all the other species this position is an aspartate. This analysismay indicate potentially critical residues in pKal that interact withX81-B01.

Example 10: In Vitro and In Vivo Assays to Assess Efficacy of a PlasmaKallikrein Binding Protein

Binding to prekallikrein vs. kallikrein: The advantage of an antibodyinhibitor of pKal that does not bind prekallikrein over an antibody thatbinds prekallikrein can be demonstrated experimentally. For example, anin vitro experiment can be designed to compare the potency of a pKalantibody inhibitor that does not bind prekallikrein (e.g. DX-2922) withone that binds prekallikrein (e.g. M6-D09) using an activated partialthromboplastin time (APTT) plasma clotting time assay. The APTT assayinduces clotting in plasma by the addition of a reagent thatspecifically activates the contact system component of the intrinsiccoagulation pathway, of which the activity of pKal is involved. It iswell known in the literature that the inhibition of pKal or that agenetic deficiency in pKal leads to prolonged aPPT (see e.g., Morishita,H., et al., Thromb Res, 1994. 73(3-4): p. 193-204; Wynne Jones, D., etal., Br J Haematol, 2004. 127(2): p. 220-3). An in vitro experiment canbe performed to measure the effect of spiking citrated human plasma withdifferent concentrations of either M6-D09 or DX-2922 on observedclotting times induced using commercially available APTT reagents and acoagulation analyzer (Table 13). It is expected that the observed EC50for APTT prolongation of M6-D09 will be significantly higher than thatof DX-2922 due to the binding of M6-D09 to the high concentrationprekallikrein (˜500 nM) in the normal plasma sample. Efficacy of theantibody inhibitor of pKal as demonstrated by prolonging APTT supportsthe potential therapeutic use of the antibody in treating or preventingcardiovascular disease associated with aberrant clot formation, such asmay be observed in atherosclerosis, stroke, vasculitis, aneurism, andpatients implanted with ventricular assist devices.

TABLE 13 Study Design to Measure Effect of Antibody Inhibitors of pKalon APTT Condition Observed Effect on APPT No treatment, just plasmaNormal Prekallikrein depleted plasma Maximum prolongation control(commercially available) M6-D09 at low concentration Normal M6-D09 atmiddle concentration Normal M6-D09 at high concentration Prolonged APTTDX-2922 at low concentration Prolonged APTT DX-2922 at middleconcentration Prolonged APTT DX-2922 at high concentration Maximumprolongation

Efficacy in a Rat Model of Edema:

An in vivo experiment can also be conducted to demonstrate the increasedpotency of an antibody inhibitor of pKal that does not bindprekallikrein. The carrageenan-induced paw edema (CPE) model of edema inrats is a common pharmacology model. A group of rats will be treatedwith escalating doses of M6-D09 and DX-2922 by intraperitoneal (IP)injection prior to injecting carrageenan (e.g. 0.1 mL of a 10% w/vsolution) in the paws of the rats (Table 14). It is expected thatDX-2922 will be more effective in reducing the observed paw swellingthan M6-D09. Efficacy of the antibody supports the therapeutic use ofthe antibody in various inflammatory diseases that are associated witheither swelling (e.g. hereditary angioedema, stroke induced edema, brainedema) or bradykinin mediated inflammation and pain (e.g. rheumatoidarthritis, inflammatory bowel disease).

TABLE 14 Study Design to Observe Effect of Antibody Inhibitors on CPEExample Dose Group Treatment (mg/Kg) Effect Expected 1 Vehicle N/AMaximum swelling 2 Indomethacin 5 Maximum reduction of swelling(positive control) 3 M6-D09 1 No effect on swelling 4 M6-D09 3 No effecton swelling 5 M6-D09 10 Intermediate reduction of swelling 6 DX-2922 1No effect on swelling 7 DX-2922 3 Intermediate reduction of swelling 8DX-2922 10 Maximum reduction of swelling

Measuring Half-Life:

The pharmacokinetic properties of DX-2922 will be determined in rats andcynomolgus monkeys in a study design outlined in Table 15. Serum will becollected at the times indicated following IV injection of DX-2922. Theconcentration of DX-2922 will be determined by ELISA and plotted versustime in order to obtain pharmacokinetic parameters (clearance, halflife, volume of distribution, etc). It is expected that DX-2922 willhave a half-life greater than 5 days in cynomolgus monkeys that willscale to a half life greater than 7 days in humans.

TABLE 15 A Pharmacokinetic study of plasma kallikrein inhibitor pKaladministered by intravenous and subcutaneous routes to cynomolgusmonkeys. Dosage Number Level of (mg/kg/ Animals Group Treatment day)Route (sex) Serum Collection 1 pKAL 20 IV 4 (M) Pre-dose 0.08, 0.25.0.5, 1, 4, 24 hours Day 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 2pKAL 20 IV 4 (M) Pre-dose 0.08, 0.25. 0.5, 1, 4, 24 hours Day 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14

Example 11: Epitope mapping using amino acid mutations of pKal

Based on the epitope mapping studies described herein in Example 8, weinspected the published 3 dimensional model in the RCSB Protein DataBank (available on the world wide web at rcsb.org; pdb code 2ANY) andidentified a collection of sets of amino acids in surface accessibleloops near the enzyme active site that we reasoned could interact withthe antibody binding resulting in enzyme inhibition. These amino acidswere substituted for alanine and the catalytic domain of each of themutant was expressed in Pichia pastoris with a His tag fusion andpurified by IMAC. Four different mutant pKal mutants were synthesizedand tested:

Mutant 1: Amino acids S478, N481, S525, and K526 of the human kallikreinsequence (Accession No. NP_00883.2) were mutated to alanine. These aminoacids were determined to be involved in substrate recognition (S3subsite).

Mutant 2: Amino acid residues R551, Q553, Y555, T558 and R560 of thehuman kallikrein sequence (Accession No. NP_00883.2) were mutated toalanine. It was determined that these residues are involved in theactive site substrate recognition (S subsite).

Mutant 3: Amino acids D572, K575, and D577 of the human kallikreinsequence (Accession No. NP_00883.2) were mutated to alanine. These aminoacid residues are involved in substrate recognition (5 subsite)

Mutant 4: Amino acids N395, S397 and S398 of the human kallikreinsequence (Accession No. NP_00883.2) were mutated to alanine. Theseresidues are distal from the active site of plasma kallikrein.

Three of the 4 mutants (Mutant 1, 2, and 4) have similar activity tothat of the wildtype catalytic domain of pKal. The amino acidsubstitutions in Mutant 3 yielded an inactive protein that was notrecognized in SPR (Biacore) binding assays by any of the testedanti-pKal antibodies.

The antibodies tested for inhibition of mutants 1, 2 and 4 are shownherein in Table 16. Based on the measured K_(lapp) values for theantibodies in Group 1 (i.e., antibodies that inhibit human and mousepKal but do not bind prekallikrein) it is evident that this group ofantibodies binds an epitope on pKal that contains the amino acids thatwere mutated in Mutant 2 but were not dependent on residues mutated inMutants 1 or 4. In addition, the interaction of plasma kallikreinbinding proteins X81-B01/X101-A01/DX-2922 and affinity maturedderivative X115-B07 to kallikrein is adversely affected by thesubstitutions in Mutant 1. For an example of the differences in theability of the antibodies to bind prekallikrein see e.g., FIGS. 11A and11B, which compares prekallikrein the binding of DX-2922 (Group 1) tothat of M6-D09 (Group 3).

The antibodies in Group 2 (i.e., those that inhibit human pKal not mousepkal and do not bind prekallikrein) were not significantly affected bythe mutated amino acids indicating that they make contact with alternateamino acids. The Group 2 antibodies are likely to bind near the activesite, as they were unable to bind pKal complexed with a Kunitz domain(EPI-KAL2), which is known to bind at the active site of a serineprotease. Furthermore, one of the antibodies in Group 2 (M145-D11) issimilar to those in Group 1 in that it is unable to bind pKal in aBiacore assay that is inactivated with the suicide inhibitor AEBSF(4-(2-Aminoethyl) benzenesulfonyl fluoride hydrochloride), which is asmall molecule covalent inhibitor of trypsin-like serine proteases (FIG.12). However, the other antibody (M29-D09) assigned to Group 2 was ableto bind AEBSF inactivated pKal, indicating that it may bind a differentepitope than M145-D11 despite sharing similar binding properties.

The antibodies in Group 3 inhibited human and mouse pKal but boundprekallikrein. One of these antibodies, M6-D09, was unable to bind pKalinactivated by either EPI-KAL2 or AEBSF, indicating that this group ofpKal inhibitors interacts with alternative amino acids near the activesite. The K_(i,app) for M6-D09 increased approximately 5-fold towardsMutant 2 (i.e., decreased potency of M6-D09).

Example 12: Affinity Maturation

In addition to the affinity maturation described herein in Examples 4and 5, which involved optimization of the light chain we attempted tofurther optimize affinity with libraries that vary amino acids in theCDR1, CDR2, and CDR3 regions of the variable heavy chain of twodifferent parental anti-pKal antibodies. Both of the antibodies selectedfor further optimization (X63-G06 and M162-A04) exhibit desirableproperties for further development as a therapeutic antibody inhibitorof plasma kallikrein; properties which include: a) complete inhibitionof human and rodent plasma kallikrein and b) no binding toprekallikrein. In some embodiments, complete inhibition of human pKal isessential to block the activity of plasma kallikrein in disease uses.Inhibition of rodent pKal facilitates preclinical development includingtoxicity assessment. The lack of binding to prekallikrein is a highlydesirable property for an antibody inhibitor of pKal to maximize thebioavailability of the antibody therapeutic towards active pKal targetand to potentially reduce the dose required for efficacy.

Affinity maturation was performed using 4 different phage displaylibraries. For each parental antibody (e.g., I62-A04), a library wasconstructed that contained varied amino acid positions in both the CDR1and the CDR2 of the heavy chain. An additional library was constructedfor each of the two parental antibodies wherein positions in the CDR3 ofthe heavy chain were varied. Each of these 4 phage display librarieswere selected (panned) with decreasing amounts of active pKal in eachsubsequent round in order to obtain high affinity antibodies. Tominimize the appearance of prekallikrein binding in the selectedantibody output libraries were initially depleted against immobilizedprekallikrein. After screening as Fab fragments, we discovered theaffinity matured antibodies shown in Table 16 (i.e. the antibodies withthe identification number starting with “X115”).

Four discovered antibodies (X115-B07, X115-D05, X115-E09, and X115-H06)are derived from the DX-2922 parental antibody (also known as X63-G06 asa Fab fragment, X81-B01 as an IgG produced in 293T cells, or X101-A01 asan IgG produced in CHO cells) were found to be potent pKal inhibitors.For comparison the amino acid sequence of DX-2922 is shown. It isevident that three of the affinity matured antibodies (X115-B07,X115-E09, and X115-H06) contain mutations in Hv-CDR3; whereas X115-D05has a different Hv-CDR1/CDR2. Four other discovered antibodies(X115-F02, X115-A03, X115-D01, and X115-G04) are derived from theM162-A04 parental antibody. All 8 affinity matured antibodies do notbind prekallikrein.

TABLE 17(SEQ ID NOs: 1851-1934 numbered down the columns starting on the left):WT Full cat. Mu- Mu- Mu- length Do- tant tant tant com- pKal main 1 2 4com- petes Ki, Ki, Ki, Ki, Ki, petes with Iso- LV- HV- app app app appapp with epi- late CDR1 LV-CDR2 LV-CDR3 CDR1 HV-CDR2 HV-CDR3 (nM) (nM)(nM) (nM) (nM) AEBSF ka12 Group DX- RTSQFV GASSRAT QQSSRTPWT HYLMTYISPSGGHT VARGIAARSRTSY 0.2 0.22 14 20 0.25 y y 1 2922 NSNYLA IYADSVKGFDY X115- RTSQFV GASSRAT QQSSRTPWT HYLMT YISPSGGHT VGQGIRGRSRTSY 0.33 4.7 47 0.14 y nd 1 B07 NSNYLA IYADSVKG FAQ X115- RTSQFV GASSRATQQSSRTPWT DYMMA SIVPSGGHT VARGIAARSRTSY 0.25 nd nd nd nd y nd 1 D05NSNYLA HYADSVKG FDY X115- RTSQFV GASSRAT QQSSRTPWT HYLMT YISPSGGHTVAQGIAARSRTSS 0.34 nd nd nd nd y nd 1 E09 NSNYLA IYADSVKG VDQ X115-RTSQFV GASSRAT QQSSRTPWT HYLMT YISPSGGHT VAQGISARSRTSY 0.35 nd nd nd ndy nd 1 H06 NSNYLA IYADSVKG FDY M162- RASQSI KASTLES QQYNTYWT HYIMMGIYSSGGIT RRTGIPARDAFDI nd nd nd nd y y 1 A04 SSWLA VYADSVKG X115-RASQSI KASTLES QQYNTYWT HYIMM GIYSSGGIT RRIGVPARDSFDM 0.16 0.16  0.23 3.7 0.13 y nd 1 A03 SSWLA VYADSVKG X115- RASQSI KASTLES QQYNTYWT IYSMHSIYPSRGMT RRTGIPARDAFDI 0.18 0.18  0.26  2.5 0.12 y nd 1 D01 SSWLAWYADSVKG X115- RASQSI KASTLES QQYNTYWT HYIMM GIYSSGGIT RRIGVPRRDEFDI0.089 0.09  0.14  5.9 0.1 y y 1 F02 SSWLA VYADSVKG X115- RASQSI KASTLESQQYNTYWT HYIMM GIYSSGGIT RRTGVPRRDEFDI 0.6 0.3  0.4  2.2 0.3 y y 1 G04SSWLA VYADSVKG M29- SGNKLG QDTKRPS QAWDSSIV WYTMV YIYPSGGATGSYDYIWGFYSDH 0.7 0.24  0.27  0.34 0.39 n y 2 D09 DKYVA I FYADSVKG M145-SGDKLG QDIKRPS QAWDSPNA HYRMS SIYPSGGRT DKFEWRLLFRGIG 0.79 0.16  0.23 0.1 0.21 y y 2 D11 DKYTS RV VYADSVKG NDAFDI M06- RASQSI AASTLQSQQLSGYPH FYYMV VIYPSGGIT DKWAVMPPYYYYA 5.9 2.5  3.4 13.5 2.9 y y 3 D09RNYLN T VYADSVKG MDV M35- RASQSV DASNRAT QQRSNWPR YYHMS VISPSGGSTGGSSDYAWGSYRR 2.9 0.8  0.09  1.1 0.8 nd nd 3 G04 SSYLA GFT KYADSVKGPYYFDY

Equilibrium K_(i,app) measurements. Apparent Inhibition constants(K_(i,app) values) were measured by pre-incubating enzyme and inhibitorsolutions prior to initiating the reactions with substrate. Enzyme andinhibitor were pre-incubated for 2 hours at 30° C. in a 96-well plate byadding 10 μL of a 10× enzyme solution and 10 μL of 10× inhibitorsolutions to 70 μL of reaction buffer. Reactions were initiated by theaddition of 10 μL of a 10× concentrated stock of substrate, and weremonitored at 30° C. in a fluorescence plate reader with the excitationand emission wavelengths set at 360 nm/460 nm, respectively. Kineticdata were acquired by the increase in fluorescence, and initial ratesfor each condition were plotted against the total inhibitorconcentration. The data was fit to the following equation for tightbinding inhibitors:

$\begin{matrix}{A = {A_{0} - {A_{inh}\left( \frac{\left( {K_{i},_{app}{{+ {Inh}} + E}} \right) - \sqrt{\left( {K_{i,{app}} + {Inh} + E} \right)^{2} - {4 \cdot {Inh} \cdot E}}}{2 \cdot E} \right)}}} & {{Eqn}.\mspace{14mu} 1}\end{matrix}$

Where A=initial rate observed at each inhibitor concentration;A_(o)=initial rate observed in the absence of inhibitor; A_(inh)=initialrate observed for the enzyme inhibitor complex; Inh=concentration ofinhibitor; E=total enzyme concentration (treated as a floatedparameter); and K_(i,app)=apparent equilibrium inhibition constant.

Groups of Antibody Inhibitors.

Antibodies in Group 1 inhibit human and mouse pKal but do not bindprekallikrein. Antibodies in Group 2 inhibit human but not mouse pKaland do not bind prekallikrein. Antibodies in Group 3 inhibit human andmouse pKal but bind prekallikrein.

Biacore Competition Analysis with an Exemplary Kallikrein Antibody,Epi-Kal2.

Epi-Kal2 is an antibody inhibitor of kallikrein that acts by binding tothe active site of kallikrein (for sequence see Example 8). The Biacorecompetition analysis is used herein as an assay to determine whether atest kallikrein antibody binds to the same site as epi-Kal2 and isassessed by measuring the competition (e.g., displacement) betweenepi-Kal2 and the test antibody for binding to the active site.

Goat anti-human Fc fragment specific IgG or anti-human Fab IgG wasimmobilized by amine coupling on a CM5 sensor chip at immobilizationdensities of approximately 5000 RU. Anti-pKal antibodies or sFabs werecaptured on their respective surfaces by injecting a 50 nM solution ofIgG/sFab for 1-2 minutes at 5 at μl/min. Human pKal (100 nM) or humanpKal-ep-kal2 complex (100 nM hpKal that had been pre-incubated with 1 μMepi-kal2 for 1 hour at room temperature) were injected over the capturedIgGs or sFabs for 5 minutes at 20-50 μl/min followed by a 5-10 minutedissociation phase. Binding responses were recorded at the end of theassociation phase. Anti-pKal IgGs or sFabs were considered to competewith epi-kal2 for binding to human pKal if binding of the pKal-epi-kal2complex to anti-pKal antibodies was significantly reduced (>70%) ascompared to an injection of hpKal only. The sensor chip surface wasregenerated with a pulse of 10 mM glycine pH 1.5 at a flow rate of 100μmin. Measurements were performed at 25° C. using HBS-P (10 mM HEPES pH7.4, 150 mM NaCl and 0.005% surfactant P20) as the running buffer.Results from the Biacore competition analysis for epi-Kal2 are shownherein in FIGS. 11A and 11B.

Biacore Competition Analysis with the Small Molecule KallikreinInhibitor, AEBSF.

AEBSF (i.e., 4-(2-aminoethyl)benzene sulfonyl fluoride hydrochloride) isa small molecule inhibitor of kallikrein. The Biacore competitionanalysis is used herein to determine whether a test antibody binds tothe same site (or an overlapping site) utilized by AEBSF for kallikreininhibition.

Goat anti-human Fc fragment specific IgG or anti-human Fab IgG wasimmobilized by amine coupling on a CM5 sensor chip at immobilizationdensities of approximately 5000 RU. Anti-pKal IgGs or sFabs werecaptured on their respective surfaces by injecting a 50 nM solution ofIgG/sFab for 1-2 minutes at 5 at μl/min. Human pKal (100 nM) or humanpKal-AEBSF complex (100 nM hpKal that had been pre-treated with 1 mMAEBSF for 1 hour at room temperature) were injected over the capturedIgGs or sFabs for 5 minutes at 20-50 μl/min followed by a 5-10 minutedissociation phase. Binding responses were recorded at the end of theassociation phase. Anti-pKal IgGs or sFabs were considered to competewith AEBSF for binding to human pKal if binding of the pKal-AEBSFcomplex to anti-pKal antibodies was significantly reduced (>70%) ascompared to an injection of hpKal only. The sensor chip surface wasregenerated with a pulse of 10 mM glycine pH 1.5 at a flow rate of 100μmin. Measurements were performed at 25° C. using HBS-P (10 mM HEPES pH7.4, 150 mM NaCl and 0.005% surfactant P20) as the running buffer.Results from the Biacore competition analysis for AEBSF are shown hereinin FIG. 12.

The following are Sequences for the light chain variable regions (LV),and heavy chain variable regions (HV) regions for 8 Exemplary AffinityMatured Anti-pKal Antibodies (SEQ ID NOs:1935-1990):

559A-M0029-D09-LVQSALTQPPTVSVSPGQTARITCSGNKLGDKYVAWYQQKPGQSPMLVIYQDTKRPSRVSERFSGSNSANTATLSISGTQALDEADYYCQAWDSSIVIFGGGTRLTVL 559A-M0145-D11-LVQSVLTQPPSVSVSPGQTASITCSGDKLGDKYTSWYQQRPGQSPVLVIYQDIKRPSGIPERFSGSNSGNTATLTISGTQAMDEADYYCQAWDSPNARVFGSGTKVTVL 559A-M0162-A04-LVDIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPNLLIYKASTLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYNTYWTFGQGTKVEIK 559A-X0101-A01-LVEIVLTQSPGTLSLSPGERATLSCRTSQFVNSNYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQSSRTPWTFGQGTKVEIK 559A-X0115-A03-LVDIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYKASTLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYNTYWTFGQGTKVEIK 559A-X0115-B07-LVEIVLTQSPGTLSLSPGERATLSCRTSQFVNSNYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQSSRTPWTFGQGTKVEIK 559A-X0115-D01-LVDIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYKASTLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYNTYWTFGQGTKVEIK 559A-X0115-D05-LVEIVLTQSPGTLSLSPGERATLSCRTSQFVNSNYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQSSRTPWTFGQGTKVEIK 559A-X0115-E09-LVEIVLTQSPGTLSLSPGERATLSCRTSQFVNSNYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQSSRTPWTFGQGTKVEIK 559A-X0115-F02-LVDIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYKASTLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYNTYWTFGQGTKVEIK 559A-X0115-G04-LVDIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYKASTLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYNTYWTFGQGTKVEIK 559A-X0115-H06-LVEIVLTQSPGTLSLSPGERATLSCRTSQFVNSNYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQSSRTPWTFGQGTKVEIK 559A-M0006-D09-LVDIQMTQSPSSLSASVGDRVTITCRASQSIRNYLNWYQQKPGKAPNLLIYAASTLQSGVPARFSGSGSGTDFTLTISSLQPEDFATYYCQQLSGYPHTFGQGTKLEIK 559A-M0035-G04-LVQDIQMTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPRGFTFGPGTKVDIK 559A-M0029-D09-HVEVQLLESGGGLVQPGGSLRLSCAASGFTFSWYTMVWVRQAPGKGLEWVSYIYPSGGATFYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAMGSYDYIWGFYSDHWGQGTLVTVSS 559A-M0145-D11-HVEVQLLESGGGLVQPGGSLRLSCAASGFTFSHYRMSWVRQAPGKGLEWVSSIYPSGGRTVYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDKFEWRLLFRGIGNDAFDIWGQGTMVTVSS559A-M0162-A04-HVEVQLLESGGGLVQPGGSLRLSCAASGFTFSHYIMMWVRQAPGKGLEWVSGIYSSGGITVYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAYRRTGIPRRDAFDIWGQGTMVTVSS 559A-X0101-A01-HVEVQLLESGGGLVQPGGSLRLSCAASGFTFSHYLMTWVRQAPGKGLEWVSYISPSGGHTIYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARVARGIAARSRTSYFDYWGQGTLVTVSS 559A-X0115-A03-HVEVQLLESGGGLVQPGGSLRLSCAASGFTFSHYIMMWVRQAPGKGLEWVSGIYSSGGITVYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAWRRIGVPRRDSFDMWGQGTMVTVSS 559A-X0115-B07-HVEVQLLESGGGLVQPGGSLRLSCAASGFTFSHYLMTWVRQAPGKGLEWVSYISPSGGHTIYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAMVGQGIRGRSRTSYFAQWGQGTLVTVSS 559A-X0115-D01-HVEVQLLESGGGLVQPGGSLRLSCAASGFTFSIYSMHWVRQAPGKGLEWVSSIYPSRGMTWYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAYRRTGIPRRDAFDIWGQGTMVTVSS 559A-X0115-D05-HVEVQLLESGGGLVQPGGSLRLSCAASGFTFSDYMMAWVRQAPGKGLEWVSSIVPSGGHTHYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARVARGIAARSRTSYFDYWGQGTLVTVSS 559A-X0115-E09-HVEVQLLESGGGLVQPGGSLRLSCAASGFTFSHYLMTWVRQAPGKGLEWVSYISPSGGHTIYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARVAQGIAARSRTSSVDQWGQGTLVTVSS 559A-X0115-F02-HVEVQLLESGGGLVQPGGSLRLSCAASGFTFSHYIMMWVRQAPGKGLEWVSGIYSSGGITVYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAYRRIGVPRRDEFDIWGQGTMVTVSS 559A-X0115-G04-HVEVQLLESGGGLVQPGGSLRLSCAASGFTFSHYIMMWVRQAPGKGLEWVSGIYSSGGITVYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAYRRTGVPRRDEFDIWGQGTMVTVSS 559A-X0115-H06-HVEVQLLESGGGLVQPGGSLRLSCAASGFTFSHYLMTWVRQAPGKGLEWVSYISPSGGHTIYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARVAQGISARSRTSYFDYWGQGTLVTVSS 559A-M0006-D09-HVEVQLLESGGGLVQPGGSLRLSCAASGFTFSFYYMVWVRQAPGKGLEWVSVIYPSGGITVYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDKWAVMPPYYYYAMDVWGQGTTVTVSS 559A-M0035-G04-HVEVQLLESGGGLVQPGGSLRLSCAASGFTFSYYHMSWVRQAPGKGLEWVSVISPSGGSTKYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGGSSDYAWGSYRRPYYFDYWGQGTLVTVSS559A-M0029-D09 LVCAGAGCGCTTTGACTCAGCCACCCACAGTGTCTGTGTCCCCAGGACAGACAGCCAGGATCACCTGCTCTGGAAATAAATTGGGGGATAAATATGTTGCCTGGTATCAGCAGAAGCCAGGCCAGTCCCCTATGTTGGTCATCTATCAAGATACTAAGCGCCCCTCAAGAGTTTCTGAGCGATTCTCTGGCTCCAACTCTGCGAATACAGCCACTCTGTCCATCAGCGGGACCCAGGCTCTGGATGAGGCTGACTATTACTGTCAGGCGTGGGACAGCAGCATTGTGATCTTCGGCGGAGGGACCAGGCTGACCGTCCTA 559A-M0145-D11 LVCAGAGCGTCTTGACTCAGCCACCCTCAGTGTCCGTGTCTCCAGGACAGACAGCCAGCATCACCTGCTCTGGAGATAAATTGGGGGATAAATATACTTCCTGGTATCAGCAGAGGCCAGGCCAGTCCCCTGTATTGGTCATCTATCAAGATATCAAGCGGCCCTCAGGGATCCCTGAGCGATTCTCTGGCTCCAACTCTGGGAACACAGCCACTCTGACCATCAGCGGGACCCAGGCTATGGATGAGGCTGACTATTACTGTCAGGCGTGGGACAGTCCCAATGCGAGGGTCTTCGGATCTGGGACCAAGGTCACCGTCCTA 559A-M0162-A04 LVGACATCCAGATGACCCAGTCTCCTTCCACCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCCAGTCAGAGTATCAGTAGTTGGTTGGCCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAACCTCCTGATCTATAAGGCGTCTACTTTAGAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGAATTCACTCTCACCATCAGCAGCCTGCAGCCTGATGATTTTGCAACTTATTACTGCCAACAGTATAATACTTATTGGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAA 559A-X0101-A01 LVGAGATCGTGCTGACCCAGTCCCCTGGCACCCTGTCTCTGTCTCCCGGCGAGAGAGCCACCCTGTCCTGCCGGACCTCCCAGTTCGTGAACTCCAACTACCTGGCTTGGTATCAGCAGAAGCCAGGCCAGGCCCCTAGACTGCTGATCTACGGCGCCTCTTCCAGAGCCACCGGCATCCCTGACCGGTTCTCCGGCTCTGGCTCCGGCACCGACTTCACCCTGACCATCTCCCGGCTGGAACCTGAGGACTTCGCCGTGTACTACTGCCAGCAGTCCTCCCGGACCCCTTGGACCTTTGGCCAGGGCACCAAGGTGGAGATCAAG 559A-X0115-A03 LVGACATCCAGATGACCCAGTCCCCCTCCACCCTGTCCGCCTCTGTGGGCGACAGAGTGACCATCACCTGTCGGGCCTCCCAGTCCATCTCCAGCTGGCTGGCCTGGTATCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATCTACAAGGCCAGCACCCTGGAATCCGGCGTGCCCTCCAGATTCTCCGGCTCTGGCTCCGGCACCGAGTTCACCCTGACCATCAGCTCCCTGCAGCCCGACGACTTCGCCACCTACTACTGCCAGCAGTACAACACCTACTGGACCTTCGGCCAGGGCACCAAGGTGGAAATCAAG 559A-X0115-B07 LVGAGATCGTGCTGACCCAGTCCCCTGGCACCCTGTCTCTGTCTCCCGGCGAGAGAGCCACCCTGTCCTGCCGGACCTCCCAGTTCGTGAACTCCAACTACCTGGCTTGGTATCAGCAGAAGCCAGGCCAGGCCCCTAGACTGCTGATCTACGGCGCCTCTTCCAGAGCCACCGGCATCCCTGACCGGTTCTCCGGCTCTGGCTCCGGCACCGACTTCACCCTGACCATCTCCCGGCTGGAACCTGAGGACTTCGCCGTGTACTACTGCCAGCAGTCCTCCCGGACCCCTTGGACCTTTGGCCAGGGCACCAAGGTGGAGATCAAG 559A-X0115-D01 LVGACATCCAGATGACCCAGTCCCCCTCCACCCTGTCCGCCTCTGTGGGCGACAGAGTGACCATCACCTGTCGGGCCTCCCAGTCCATCTCCAGCTGGCTGGCCTGGTATCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATCTACAAGGCCAGCACCCTGGAATCCGGCGTGCCCTCCAGATTCTCCGGCTCTGGCTCCGGCACCGAGTTCACCCTGACCATCAGCTCCCTGCAGCCCGACGACTTCGCCACCTACTACTGCCAGCAGTACAACACCTACTGGACCTTCGGCCAGGGCACCAAGGTGGAAATCAAG 559A-X0115-D05 LVGAGATCGTGCTGACCCAGTCCCCTGGCACCCTGTCTCTGTCTCCCGGCGAGAGAGCCACCCTGTCCTGCCGGACCTCCCAGTTCGTGAACTCCAACTACCTGGCTTGGTATCAGCAGAAGCCAGGCCAGGCCCCTAGACTGCTGATCTACGGCGCCTCTTCCAGAGCCACCGGCATCCCTGACCGGTTCTCCGGCTCTGGCTCCGGCACCGACTTCACCCTGACCATCTCCCGGCTGGAACCTGAGGACTTCGCCGTGTACTACTGCCAGCAGTCCTCCCGGACCCCTTGGACCTTTGGCCAGGGCACCAAGGTGGAGATCAAG 559A-X0115-E09 LVGAGATCGTGCTGACCCAGTCCCCTGGCACCCTGTCTCTGTCTCCCGGCGAGAGAGCCACCCTGTCCTGCCGGACCTCCCAGTTCGTGAACTCCAACTACCTGGCTTGGTATCAGCAGAAGCCAGGCCAGGCCCCTAGACTGCTGATCTACGGCGCCTCTTCCAGAGCCACCGGCATCCCTGACCGGTTCTCCGGCTCTGGCTCCGGCACCGACTTCACCCTGACCATCTCCCGGCTGGAACCTGAGGACTTCGCCGTGTACTACTGCCAGCAGTCCTCCCGGACCCCTTGGACCTTTGGCCAGGGCACCAAGGTGGAGATCAAG 559A-X0115-F02 LVGACATCCAGATGACCCAGTCCCCCTCCACCCTGTCCGCCTCTGTGGGCGACAGAGTGACCATCACCTGTCGGGCCTCCCAGTCCATCTCCAGCTGGCTGGCCTGGTATCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATCTACAAGGCCAGCACCCTGGAATCCGGCGTGCCCTCCAGATTCTCCGGCTCTGGCTCCGGCACCGAGTTCACCCTGACCATCAGCTCCCTGCAGCCCGACGACTTCGCCACCTACTACTGCCAGCAGTACAACACCTACTGGACCTTCGGCCAGGGCACCAAGGTGGAAATCAAG 559A-X0115-G04 LVGACATCCAGATGACCCAGTCCCCCTCCACCCTGTCCGCCTCTGTGGGCGACAGAGTGACCATCACCTGTCGGGCCTCCCAGTCCATCTCCAGCTGGCTGGCCTGGTATCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATCTACAAGGCCAGCACCCTGGAATCCGGCGTGCCCTCCAGATTCTCCGGCTCTGGCTCCGGCACCGAGTTCACCCTGACCATCAGCTCCCTGCAGCCCGACGACTTCGCCACCTACTACTGCCAGCAGTACAACACCTACTGGACCTTCGGCCAGGGCACCAAGGTGGAAATCAAG 559A-X0115-H06 LVGAGATCGTGCTGACCCAGTCCCCTGGCACCCTGTCTCTGTCTCCCGGCGAGAGAGCCACCCTGTCCTGCCGGACCTCCCAGTTCGTGAACTCCAACTACCTGGCTTGGTATCAGCAGAAGCCAGGCCAGGCCCCTAGACTGCTGATCTACGGCGCCTCTTCCAGAGCCACCGGCATCCCTGACCGGTTCTCCGGCTCTGGCTCCGGCACCGACTTCACCCTGACCATCTCCCGGCTGGAACCTGAGGACTTCGCCGTGTACTACTGCCAGCAGTCCTCCCGGACCCCTTGGACCTTTGGCCAGGGCACCAAGGTGGAGATCAAG 559A-M0006-D09 LVGACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGTATTCGCAACTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAACCTCCTGATCTATGCTGCATCCACTTTGCAAAGTGGGGTCCCAGCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACTATCAGCAGTCTGCAGCCTGAAGATTTTGCAACTTATTACTGTCAACAGCTTAGTGGTTACCCCCACACTTTTGGCCAGGGGACCAAGCTGGAGATCAAA 559A-M0035-G04 LVCAAGACATCCAGATGACCCAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCTACTTAGCCTGGTACCAACAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGATGCATCCAACAGGGCCACTGGCATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGCCTAGAGCCTGAAGATTTTGCAGTTTATTACTGTCAGCAGCGTAGCAACTGGCCTCGCGGATTCACTTTCGGCCCTGGGACCAAAGTGGATATCAAA 559A-M0029-D09 HVGAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTCTTGCGCTGCTTCCGGATTCACTTTCTCTTGGTACACTATGGTTTGGGTTCGCCAAGCTCCTGGTAAAGGTTTGGAGTGGGTTTCTTATATCTATCCTTCTGGTGGCGCTACTTTTTATGCTGACTCCGTTAAAGGTCGCTTCACTATCTCTAGAGACAACTCTAAGAATACTCTCTACTTGCAGATGAACAGCTTAAGGGCTGAGGACACGGCCGTGTATTACTGTGCGATGGGTTCATATGATTACATTTGGGGATTTTATAGTGACCACTGGGGCCAGGGAACCCTGGTCACCGTCTCAAGC 559A-M0145-D11 HVGAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTCTTGCGCTGCTTCCGGATTCACTTTCTCTCATTACCGTATGTCTTGGGTTCGCCAAGCTCCTGGTAAAGGTTTGGAGTGGGTTTCTTCTATCTATCCTTCTGGTGGCCGTACTGTTTATGCTGACTCCGTTAAAGGTCGCTTCACTATCTCTAGAGACAACTCTAAGAATACTCTCTACTTGCAGATGAACAGCTTAAGGGCTGAGGACACGGCCGTGTATTACTGTGCGAAAGATAAGTTCGAGTGGAGGTTATTATTTCGCGGGATTGGAAATGATGCTTTTGATATCTGGGGCCAAGGGACAATGGTCACCGTCTCAAGC 559A-M0162-A04 HVGAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTCTTGCGCTGCTTCCGGATTCACTTTCTCTCATTACATTATGATGTGGGTTCGCCAAGCTCCTGGTAAAGGTTTGGAGTGGGTTTCTGGTATCTATTCTTCTGGTGGCATTACTGTTTATGCTGACTCCGTTAAAGGTCGCTTCACTATCTCTAGAGACAACTCTAAGAATACTCTCTACTTGCAGATGAACAGCTTAAGGGCTGAGGACACGGCCGTGTATTACTGTGCGTACCGCCGGACTGGGATTCCAAGAAGAGATGCTTTTGATATCTGGGGCCAAGGGACAATGGTCACCGTCTCAAGC 559A-X0101-A01 HVGAGGTGCAATTGCTGGAATCCGGCGGAGGTCTGGTGCAGCCTGGCGGCTCCCTGAGACTGTCTTGCGCCGCCTCCGGCTTCACCTTCTCCCACTACCTGATGACCTGGGTGCGCCAGGCTCCTGGCAAGGGCCTCGAATGGGTGTCCTACATCTCCCCCTCTGGCGGCCACACCATCTACGCCGACTCCGTGAAGGGCCGGTTCACCATCTCCCGGGACAACTCCAAGAACACCCTGTATCTGCAGATGAACTCCCTGAGGGCCGAGGACACCGCCGTGTACTACTGCGCCAGGGTGGCCAGAGGAATCGCCGCCAGGTCCCGGACCTCCTACTTCGACTACTGGGGCCAGGGCACCCTGGTGACCGTGTCCTCC 559A-X0115-A03 HVGAGGTGCAATTGCTGGAATCCGGCGGAGGACTGGTGCAGCCTGGCGGCTCCCTGAGACTGTCTTGCGCCGCCTCCGGCTTTACCTTCTCCCACTACATCATGATGTGGGTGCGACAGGCTCCAGGCAAGGGCCTGGAATGGGTGTCCGGCATCTACTCCTCCGGCGGCATCACCGTGTACGCCGACTCCGTGAAGGGCCGGTTCACCATCTCCCGGGACAACTCCAAGAACACCCTGTACCTGCAGATGAACTCCCTGCGGGCCGAGGACACCGCCGTGTACTACTGTGCCTGGCGGAGAATCGGCGTGCCCAGACGGGACTCCTTCGACATGTGGGGACAGGGCACCATGGTGACAGTGTCCTCC 559A-X0115-B07 HVGAGGTGCAATTGCTGGAATCCGGCGGAGGACTGGTGCAGCCTGGCGGCTCCCTGAGACTGTCTTGCGCCGCCTCCGGCTTCACCTTCTCCCACTACCTGATGACCTGGGTGCGACAGGCTCCTGGCAAAGGCCTGGAATGGGTGTCCTACATCTCCCCCTCTGGCGGCCACACCATCTACGCCGACTCCGTGAAGGGCCGGTTTACCATCTCCCGGGACAACTCCAAGAACACCCTGTACCTGCAGATGAACTCCCTGCGGGCCGAGGACACCGCCGTGTACTACTGTGCCATGGTCGGCCAGGGAATCCGGGGCAGATCCCGGACCTCCTACTTCGCCCAGTGGGGCCAGGGCACCCTGGTGACAGTGTCCTCT 559A-X0115-D01 HVGAGGTGCAATTGCTGGAATCCGGCGGAGGACTGGTGCAGCCTGGCGGCTCCCTGAGACTGTCTTGCGCCGCCTCCGGCTTCACCTTCTCCATCTACTCCATGCACTGGGTGCGACAGGCTCCAGGCAAGGGCCTGGAATGGGTGTCCTCCATCTACCCCTCCCGGGGCATGACTTGGTACGCCGACTCCGTGAAGGGCCGGTTCACAATCTCCCGGGACAACTCCAAGAACACCCTGTACCTGCAGATGAACTCCCTGCGGGCCGAGGACACCGCCGTGTACTACTGCGCCTACCGGCGGACCGGCATCCCTAGACGGGACGCCTTCGACATCTGGGGGCAGGGCACCATGGTGACAGTGTCCTCC 559A-X0115-D05 HVGAGGTGCAATTGCTGGAATCCGGCGGTGGACTGGTGCAGCCTGGCGGCTCCCTGAGACTGTCTTGCGCCGCCTCCGGCTTCACCTTCTCCGACTACATGATGGCCTGGGTGCGACAGGCCCCTGGCAAGGGACTGGAATGGGTGTCCTCCATCGTGCCCTCTGGCGGCCACACCCACTACGCCGACTCCGTGAAGGGCCGGTTCACCATCTCCCGGGACAACTCCAAGAACACCCTGTACCTGCAGATGAACTCCCTGCGGGCCGAGGACACCGCCGTGTACTACTGCGCCAGAGTGGCCAGAGGAATCGCCGCCAGATCCCGGACCTCCTACTTCGACTACTGGGGCCAGGGCACCCTGGTGACAGTGTCCTCC 559A-X0115-E09 HVGAGGTGCAATTGCTGGAATCCGGCGGAGGACTGGTGCAGCCTGGCGGCTCCCTGAGACTGTCTTGCGCCGCCTCCGGCTTCACCTTCTCCCACTACCTGATGACCTGGGTGCGACAGGCTCCTGGCAAAGGCCTGGAATGGGTGTCCTACATCTCCCCCTCTGGCGGCCACACCATCTACGCCGACTCCGTGAAGGGCCGGTTTACCATCTCCCGGGACAACTCCAAGAACACCCTGTACCTGCAGATGAACTCCCTGCGGGCCGAGGACACCGCCGTGTACTACTGTGCCCGGGTGGCCCAGGGAATCGCCGCCAGATCCCGGACCTCCTCTGTGGATCAGTGGGGCCAGGGCACCCTGGTGACAGTGTCCTCT 559A-X0115-F02 HVGAGGTGCAATTGCTGGAATCCGGCGGAGGACTGGTGCAGCCTGGCGGCTCCCTGAGACTGTCTTGCGCCGCCTCCGGCTTCACCTTCTCCCACTACATCATGATGTGGGTGCGACAGGCTCCTGGCAAGGGGCTGGAATGGGTGTCCGGCATCTACTCCTCCGGCGGCATCACCGTGTACGCCGACTCCGTGAAGGGCCGGTTCACCATCTCTCGGGACAACTCCAAGAACACCCTGTACCTGCAGATGAACTCCCTGCGGGCCGAGGACACCGCCGTGTACTACTGCGCCTACCGGCGGATCGGCGTGCCCAGACGGGACGAGTTCGACATCTGGGGGCAGGGCACCATGGTGACAGTGTCCTCC 559A-X0115-G04 HVGAGGTGCAATTGCTGGAATCCGGCGGAGGACTGGTGCAGCCTGGCGGCTCCCTGAGACTGTCTTGCGCCGCCTCCGGCTTCACCTTCTCTCACTACATTATGATGTGGGTGCGACAGGCTCCTGGCAAAGGCCTGGAATGGGTGTCCGGCATCTACTCCTCCGGCGGCATCACCGTGTACGCCGACTCCGTGAAGGGCCGGTTCACCATCTCCCGGGACAACTCCAAGAACACCCTGTACCTGCAGATGAACTCCCTGCGGGCCGAGGACACCGCCGTGTACTACTGCGCCTACAGACGGACCGGCGTGCCCAGACGGGACGAGTTCGATATCTGGGGGCAGGGCACCATGGTGACAGTGTCCTCC 559A-X0115-H06 HVGAGGTGCAATTGCTGGAATCCGGCGGAGGACTGGTGCAGCCTGGCGGCTCCCTGAGACTGTCTTGCGCCGCCTCCGGCTTCACCTTCTCCCACTACCTGATGACCTGGGTGCGACAGGCTCCTGGCAAAGGCCTGGAATGGGTGTCCTACATCTCCCCCTCTGGCGGCCACACCATCTACGCCGACTCCGTGAAGGGCCGGTTTACCATCTCCCGGGACAACTCCAAGAACACCCTGTACCTGCAGATGAACTCCCTGCGGGCCGAGGACACCGCCGTGTACTACTGTGCCCGGGTGGCCCAGGGAATCTCCGCCAGATCCCGGACCTCCTACTTCGATTACTGGGGCCAGGGCACCCTGGTGACAGTGTCCTCT 559A-M0006-D09 HVGAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTCTTGCGCTGCTTCCGGATTCACTTTCTCTTTTTACTATATGGTTTGGGTTCGCCAAGCTCCTGGTAAAGGTTTGGAGTGGGTTTCTGTTATCTATCCTTCTGGTGGCATTACTGTTTATGCTGACTCCGTTAAAGGTCGCTTCACTATCTCTAGAGACAACTCTAAGAATACTCTCTACTTGCAGATGAACAGCTTAAGGGCTGAGGACACGGCCGTGTATTACTGTGCGAGAGATAAATGGGCGGTGATGCCCCCCTACTACTACTACGCTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCAAGC 559A-M0035-G04 HVGAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTCTTGCGCTGCTTCCGGATTCACTTTCTCTTATTACCATATGTCTTGGGTTCGCCAAGCTCCTGGTAAAGGTTTGGAGTGGGTTTCTGTTATCTCTCCTTCTGGTGGCTCTACTAAGTATGCTGACTCCGTTAAAGGTCGCTTCACTATCTCTAGAGACAACTCTAAGAATACTCTCTACTTGCAGATGAACAGCTTAAGGGCTGAGGACACTGCAGTCTACTATTGTGCGAGAGGCGGTTCGAGCGATTACGCTTGGGGGAGTTATCGTCGACCCTACTACTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCAAGC

1.-24. (canceled)
 25. An isolated monoclonal antibody, which binds humanplasma kallikrein (pKal), wherein the antibody binds an epitope thatcomprises one or more amino acids from CLIPS peptides C1, C2, C3, C4,C5, C6, and/or C7.
 26. The isolated monoclonal antibody of claim 25,wherein the antibody binds an epitope that includes one or more aminoacids from at least two of the CLIPS peptides.
 27. The isolatedmonoclonal antibody of claim 25, wherein the antibody binds a fragmentof the pKal selected from the group consisting of: (i)AHCFDGLPLQDVWRIY (C1), (ii) TPFSQIKEIIIHQNY (C2), (iii) HDIALIKL (C3),(iv) WVTGWGFSKEKGEI (C4), (v) LVTNEECQKRYQDYKITQQ (C5), (vi)ACKGDSGGPL (C6), and (vii) SWGDI (C7).


28. The isolated monoclonal antibody of claim 25, wherein the monoclonalantibody binds human plasma prekallikrein less effectively relative tothe human pKal by a factor of at least 10 fold.
 29. The isolatedmonoclonal antibody of claim 28, which binds human plasma prekallikreinless effectively relative to the human pKal by a factor of at least 100fold.
 30. The isolated monoclonal antibody of claim 29, which bindshuman plasma prekallikrein less effectively relative to the human pKalby a factor of at least 1000 fold.
 31. The isolated monoclonal antibodyof claim 25, which competes with epi-Kal2 for binding to the human pKal.32. The isolated monoclonal antibody of claim 25, which competes withAEBSF for binding to the human pKal.
 33. The isolated monoclonalantibody of claim 25, which decreases the catalytic activity of thehuman pKal as compared to the catalytic activity of the human pKal inthe absence of the monoclonal antibody and under the same conditions.34. The isolated monoclonal antibody of claim 33, wherein the monoclonalantibody decreases the production of Factor XIIa or bradykinin by atleast 50% as compared to the production of Factor XIIa or bradykinin inthe absence of the monoclonal antibody and under the same conditions.35. The isolated monoclonal antibody of claim 34, wherein the monoclonalantibody decreases the production of Factor XIIa or bradykinin by atleast 75% as compared to the production of Factor XIIa or bradykinin inthe absence of the monoclonal antibody and under the same conditions.36. The isolated monoclonal antibody of claim 35, wherein the monoclonalantibody decreases the production of Factor XIIa or bradykinin by atleast 90% as compared to the production of Factor XIIa or bradykinin inthe absence of the monoclonal antibody and under the same conditions.37. The isolated monoclonal antibody of claim 25, which is a humanantibody.
 38. The isolated monoclonal antibody of claim 25, which is anIgG molecule.
 39. The isolated monoclonal antibody of claim 38, which isan IgG1 molecule.
 40. The isolated monoclonal antibody of claim 37,which is a germlined antibody.
 41. The isolated monoclonal antibody ofclaim 25, which is substantially a full-length antibody.
 42. Theisolated monoclonal antibody of claim 25, which comprises anantigen-binding fragment of a full-length antibody.
 43. The isolatedmonoclonal antibody of claim 25, which comprises a heavy chain variableregion and a light chain variable region.
 44. A pharmaceuticalcomposition, comprising the monoclonal antibody of claim 25 and apharmaceutically acceptable carrier.
 45. A method for treatinghereditary angioedema, comprising administering to a subject in needthereof an effective amount of the monoclonal antibody of claim 25.